afimoxifene and Inflammation

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Breast Neoplasms; Cell Proliferation; Cells, Cultured; Estrogen Receptor alpha; Estrogen Receptor Antagonists; Female; Humans; Inflammation; Ligands; Macrophages; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Molecular; Molecular Structure; NF-kappa B; Protein Conformation; Resveratrol; Signal Transduction; Structure-Activity Relationship; Sulfones

Estrogen, the female sex hormone, is known to exert anti-inflammatory and anti-atherogenic effects. Traditionally, estrogen effects were believed to be largely mediated through the classical estrogen receptors (ERs). However, there is increasing evidence that G-protein coupled receptor 30 (GPR30), a novel estrogen receptor, can mediate many estrogenic effects on the vasculature. Despite this, the localization and functional significance of GPR30 in the human vascular endothelium remains poorly understood. Given this background, we examined the subcellular location and potential anti-inflammatory roles of GPR30 using human umbilical vein endothelial cells as a model system. Inflammatory changes were induced by treatment with tumor necrosis factor (TNF), a pro-inflammatory cytokine involved in atherogenesis and many other inflammatory conditions. We found that GPR30 was located predominantly in the endothelial cell nuclei. Treatment with the selective GPR30 agonist G-1 partially attenuated the TNF induced upregulation of pro-inflammatory proteins such as intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was completely abolished by the selective GPR30 antagonist G-15, suggesting that it was indeed mediated in a GPR30 dependent manner. Interestingly, estrogen alone had no effects on TNF-treated endothelium. Concomitant activation of the classical ERs blocked the anti-inflammatory effects of G-1, indicating opposing effects of GPR30 and the classical ERs. Our findings demonstrate that endothelial GPR30 is a novel regulator of the inflammatory response which could be a potential therapeutic target against atherosclerosis and other inflammatory diseases.

Topics: Anti-Inflammatory Agents; Cell Nucleus; Cyclopentanes; Endothelium, Vascular; Estradiol; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Intercellular Adhesion Molecule-1; NF-kappa B; Quinolines; Receptors, Estrogen; Receptors, G-Protein-Coupled; Tamoxifen; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature