RESUMO
The growing epidemic of obesity and metabolic diseases calls for a better understanding of adipocyte biology. The regulation of transcription in adipocytes is particularly important, as it is a target for several therapeutic approaches. Transcriptional outcomes are influenced by both histone modifications and transcription factor binding. Although the epigenetic states and binding sites of several important transcription factors have been profiled in the mouse 3T3-L1 cell line, such data are lacking in human adipocytes. In this study, we identified H3K56 acetylation sites in human adipocytes derived from mesenchymal stem cells. H3K56 is acetylated by CBP and p300, and deacetylated by SIRT1, all are proteins with important roles in diabetes and insulin signaling. We found that while almost half of the genome shows signs of H3K56 acetylation, the highest level of H3K56 acetylation is associated with transcription factors and proteins in the adipokine signaling and Type II Diabetes pathways. In order to discover the transcription factors that recruit acetyltransferases and deacetylases to sites of H3K56 acetylation, we analyzed DNA sequences near H3K56 acetylated regions and found that the E2F recognition sequence was enriched. Using chromatin immunoprecipitation followed by high-throughput sequencing, we confirmed that genes bound by E2F4, as well as those by HSF-1 and C/EBPα, have higher than expected levels of H3K56 acetylation, and that the transcription factor binding sites and acetylation sites are often adjacent but rarely overlap. We also discovered a significant difference between bound targets of C/EBPα in 3T3-L1 and human adipocytes, highlighting the need to construct species-specific epigenetic and transcription factor binding site maps. This is the first genome-wide profile of H3K56 acetylation, E2F4, C/EBPα and HSF-1 binding in human adipocytes, and will serve as an important resource for better understanding adipocyte transcriptional regulation.
Assuntos
Adipócitos/metabolismo , Genoma Humano/genética , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Acetilação , Adipócitos/citologia , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Fator de Transcrição E2F4/genética , Fator de Transcrição E2F4/metabolismo , Perfilação da Expressão Gênica , Fatores de Transcrição de Choque Térmico , Humanos , Lisina/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fatores de Transcrição/genéticaRESUMO
INTRODUCTION: CD44 is a cell adhesion molecule believed to play a critical role in T cell and monocyte infiltration in the inflammatory process. The reduction of CD44 expression or its ability to properly interact with its key ligand, hyaluronic acid (HA), inhibits migration and subsequent activation of cells within sites of inflammation. CD44-deficient mice exhibit decreased disease in a mouse arthritis model. METHODS: Accordingly, we developed PF-03475952, a fully human IgG2 anti-CD44 monoclonal antibody (mAb). RESULTS: Binding of PF-03475952 to CD44 inhibits binding of HA and induces loss of CD44 from the cell surface. PF-03475952 also passed a series of safety pharmacology assays designed to assess the risk of the mAb to bind Fc gamma receptors, stimulate cytokine release from human whole blood, and stimulate cytokine release from peripheral blood mononuclear cells (PBMC) using plate-bound antibodies. The latter assay was designed specifically to evaluate the risk of cytokine storm that had been observed with TGN1412 (immunostimulatory CD28 superagonist mAb). PF-003475952 exhibits high-affinity binding to both human and cynomolgus monkey CD44, but does not cross-react with rodent CD44. Thus, a rat anti-mouse CD44 mAb was used to demonstrate a dose-dependent decrease of disease in mouse collagen-induced arthritis. Importantly, efficacy was correlated with >50% loss of cell surface CD44 on circulating cells. Loss of CD44 expression on CD3+ lymphocytes was monitored following a single dose of PF-03475952 in cynomolgus monkeys as a pharmacodynamic marker. The recovery of CD44 expression was found to be dose-dependent. PF-03475952 doses of 1, 10, and 100 mg/kg reduced CD44 expression below 50% for 218, 373, and >504 hours, respectively. CONCLUSION: Targeting of CD44 is a unique mechanism of action in the treatment of inflammatory diseases and is expected to reduce joint damage induced by inflammatory mediators, resulting in disease modification in inflammatory diseases such as rheumatoid arthritis.
Assuntos
Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Artrite Experimental/tratamento farmacológico , Receptores de Hialuronatos/imunologia , Imunoglobulina G/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/uso terapêutico , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Imunoglobulina G/uso terapêutico , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos DBA , Ativação Plaquetária/efeitos dos fármacos , Ligação ProteicaRESUMO
Mitogen-activated protein kinase (MAPK) pathways are implicated in joint destruction in rheumatoid arthritis (RA) by modulating the production and functions of inflammatory cytokines. Although p38 MAPK (p38) participates in signaling cascades leading to osteolysis in arthritis, the mechanisms of its action in this process remain incompletely understood. Here, we found that the osteoclast (Ocl) precursors expressed p38alpha, but not p38beta, p38delta, and p38gamma isoforms. Treatment of these cells with receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) resulted in p38 activation. Importantly, Ocl development induced by RANKL or RANKL and tumor necrosis factor (TNF)-alpha was blocked with the novel p38 inhibitor 4-(3-(4-chlorophenyl)-5-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)pyrimidine (SC-409). To validate in vitro data, p38 role was further investigated in streptococcal cell wall (SCW)-induced arthritis in rats. We found that SCW-induced joint swelling and bone destruction were attenuated by SC-409. Mechanistically, the data show that SCW-stimulated DNA binding activity of the transcription factor myocyte-enhancing factor 2 C, which is downstream of p38, was inhibited by SC-409. In addition, SC-409 inhibited SCW-stimulated expression of numerous factors, including TNF-alpha, interleukin-1beta, and RANKL. Although c-Jun NH2-terminal kinase and NF-kappaB pathways were activated in vitro by RANKL and in vivo by SCW, SC-409 had no significant effect on these pathways. In conclusion, our data show that p38 modulates the production and signaling of cytokines, thus providing a mechanism of the bone-sparing effect of SC-409 in rat arthritis. These data present SC-409 as a novel potent p38 inhibitor and suggest that p38-based therapies may be beneficial in preventing bone loss associated with RA.
Assuntos
Artrite Experimental/prevenção & controle , Osteoclastos/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Artrite Experimental/enzimologia , Artrite Experimental/patologia , Ossos da Extremidade Inferior/efeitos dos fármacos , Ossos da Extremidade Inferior/enzimologia , Ossos da Extremidade Inferior/patologia , Proteínas de Transporte/farmacologia , Linhagem Celular , Citocinas/biossíntese , Feminino , Humanos , Glicoproteínas de Membrana/farmacologia , Camundongos , Estrutura Molecular , Osteoclastos/enzimologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ligante RANK , Ratos , Ratos Endogâmicos Lew , Receptor Ativador de Fator Nuclear kappa-B , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
In recent years, stem cells have been the subject of increasing scientific interest because of their utility in numerous biomedical applications. Stem cells are capable of renewing themselves; that is, they can be continuously cultured in an undifferentiated state, giving rise to more specialized cells of the human body such as heart, liver, bone marrow, blood vessel, pancreatic islet, and nerve cells. Therefore, stem cells are an important new tool for developing unique, in vitro model systems to test drugs and chemicals and a potential to predict or anticipate toxicity in humans. The following review provides an overview of the applications of stem cell technology in the area of toxicology. Specifically, this review addresses core technologies that are emerging in the field and how they could fulfill critical safety issues such as QT prolongation and hepatotoxicity, two leading causes of failures in preclinical development of new therapeutic drugs. We report how adult stem cells derived from various sources, such as human bone marrow and placenta, can potentially generate suitable models for cardiotoxicity, hepatotoxicity, genotoxicity/epigenetic and reproductive toxicology screens. Additionally, this review addresses the role and advantages of embryonic stem cells in the aforementioned models for toxicity and how genetic selection is employed to overcome major limitations to the implementation of stem cell-based in vitro models for toxicology.
Assuntos
Células-Tronco/efeitos dos fármacos , Toxicologia/métodos , Xenobióticos/toxicidade , Adulto , Células da Medula Óssea/citologia , Células Cultivadas , Dano ao DNA , Avaliação Pré-Clínica de Medicamentos/métodos , Embrião de Mamíferos/citologia , Coração/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Placenta/citologia , Reprodução/efeitos dos fármacosRESUMO
Non-steroidal anti-inflammatory drugs (NSAIDs) specifically inhibit cyclooxygenase (COX) activity and are widely used as anti-arthritics, post-surgical analgesics, and for the relief of acute musculoskeletal pain. Recent studies suggest that non-specific NSAIDs, which inhibit both COX-1 and COX-2 isoforms, delay bone healing. The objectives of this study were 2-fold; first, to measure the relative changes in the normal expression of COX-1 and COX-2 mRNAs over a 42 day period of fracture healing and second, to compare the effects of a commonly used non-specific NSAID, ketorolac, with a COX-2 specific NSAID, Parecoxib (a pro-drug of valdecoxib), on this process. Simple, closed, transverse fractures were generated in femora of male Sprague-Dawley rats weighing approximately 450 g each. Total RNA was prepared from the calluses obtained prior to fracture and at 1, 3, 5, 7, 10, 14, 21, 35 and 42 days post-fracture and levels of COX-1 and COX-2 mRNA were measured using real time PCR. While the relative levels of COX-1 mRNA remained constant over a 21-day period, COX-2 mRNA levels showed peak expression during the first 14 days of healing and returned to basal levels by day 21. Mechanical properties of the calluses were then assessed at 21 and 35 days post-fracture in untreated animals and animals treated with either ketorolac or high or low dose parecoxib. At both 21 and 35 days after fracture, calluses in the group treated with the ketorolac showed a significant reduction in mechanical strength and stiffness when compared with controls (p<0.05). At the 21-day time point, calluses of the parecoxib treated animals showed a lower mean mechanical strength than controls, but the inhibition was not statistically significant. Based on physical analysis of the bones, 3 of 12 (25%) of the ketorolac-treated and 1 of 12 (8%) of the high dose parecoxib-treated animals showed failure to unite their fractures by 21 days, while all fractures in both groups showed union by 35 days. Histological analysis at 21 days showed that the calluses in the ketorolac-treated group contained substantial amounts of residual cartilage while neither the control nor the parecoxib-treated animals showed comparable amounts of cartilage at this stage. These results demonstrate that ketorolac and parecoxib delay fracture healing in this model, but in this study daily administration of ketorolac, a non-selective COX inhibitor had a greater affect on this process. They further demonstrate that a COX-2 selective NSAID, such as parecoxib (valdecoxib), has only a small effect on delaying fracture healing even at doses that are known to fully inhibit prostaglandin production.