RESUMO
Myeloid cell mediated mechanisms regulate synovial joint inflammation. IL-34, a macrophage (Mø) growth and differentiation molecule, is markedly expressed in neutrophil and Mø-rich arthritic synovium. IL-34 engages a newly identified independent receptor, protein-tyrosine phosphatase, receptor-type, zeta (PTPRZ), that we find is expressed by Mø. As IL-34 is prominent in rheumatoid arthritis, we probed for the IL-34 and PTPRZ-dependent myeloid cell mediated mechanisms central to arthritis using genetic deficient mice in K/BxN serum-transfer arthritis. Unanticipatedly, we now report that IL-34 and PTPRZ limited arthritis as intra-synovial pathology and bone erosion were more severe in IL-34 and PTPRZ KO mice during induced arthritis. We found that IL-34 and PTPRZ: (i) were elevated, bind, and induce downstream signaling within the synovium in arthritic mice and (ii) were upregulated in the serum and track with disease activity in rheumatoid arthritis patients. Mechanistically, IL-34 and PTPRZ skewed Mø toward a reparative phenotype, and enhanced Mø clearance of apoptotic neutrophils, thereby decreasing neutrophil recruitment and intra-synovial neutrophil extracellular traps. With fewer neutrophils and neutrophil extracellular traps in the synovium, destructive inflammation was restricted, and joint pathology and bone erosion diminished. These novel findings suggest that IL-34 and PTPRZ-dependent mechanisms in the inflamed synovium limit, rather than promote, inflammatory arthritis.
Assuntos
Artrite Experimental , Artrite Reumatoide , Interleucinas , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Animais , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Proteínas de Transporte , Inflamação , Interleucinas/metabolismo , Camundongos , Camundongos Knockout , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Membrana Sinovial/metabolismoRESUMO
Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defined target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.
Assuntos
Antígenos CD/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Receptor de Insulina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Análise de Célula Única/métodos , Androstadienos/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Citocalasina B/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Genisteína/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Antagonistas da Insulina/farmacologia , Microscopia Confocal/métodos , Microscopia de Vídeo/métodos , Extratos Vegetais/farmacologia , Transporte Proteico , Trigonella , WortmaninaRESUMO
The response rates of extensively used chemotherapeutic drugs, carboplatin (Carb) or 5-fluorouracil (5-FU) are relatively disappointing because of considerable side effects associated with their high-dose regimen. In the present study, we determined whether treatment with a cholesterol depleting agent, methyl-beta-cyclodextrin (MCD), enhances the weak efficacy of low doses of Carb or 5-FU in human breast cancer cells. Data demonstrate that pretreatment with MCD significantly potentiates the cytotoxic activity of Carb and 5-FU in both MCF-7 and MDA-MB-231. Furthermore, we explored the molecular basis of enhanced cytotoxicity, and our data revealed that low-dose treatment with these drugs in MCD pretreated cells exhibited significantly decreased Akt phosphorylation, NF-kappaB activity and down-regulation in expression of anti-apoptotic protein Bcl-2. In addition, MCD pretreated cells demonstrated an increased intracellular drug accumulation as compared to cells treated with drugs alone. Taken together, our data provide the basis for potential therapeutic application of MCD in combination with other conventional cytotoxic drugs to facilitate reduction of drug dosage that offers a better chemotherapeutic approach with low toxicity.