ABSTRACT
Peroxisome proliferator-activated receptors (PPARs) are transcription factors that belong to the nuclear receptor superfamily directly modulating gene expression by binding to specific ligands. Recently, it has been reported that PPARdelta ligands play an essential role in improvement of metabolic disorders and skin disorders. We introduce an enzyme-linked immunosorbent assay (ELISA) to screen new PPARdelta ligands. This method is based on the activation mechanism of PPARdelta where the ligand binding to PPARdelta induces the interaction of the receptor with transcriptional co-activators. We optimized a simple ELISA method for screening PPARdelta ligands. Among co-activators such as SRC-1, TIF-2, and p300, PPARdelta had more strong binding with SRC-1 in an ELISA system. GW501516 and linoleic acid, the well-known ligands of PPARdelta, increased the binding between PPARdelta and co-activators in a ligand dose-dependent manner. The recruitment of co-activator SRC-1 was also more effective than those of TIF-2 and p300. We optimized and developed a novel and useful ELISA system for the mass screening of PPARdelta ligands. This screening system may be useful in the development of drugs for metabolic disorders and skin disorders.
Subject(s)
Drug Evaluation, Preclinical/methods , Enzyme-Linked Immunosorbent Assay/methods , PPAR delta/metabolism , Cloning, Molecular , E1A-Associated p300 Protein/chemistry , E1A-Associated p300 Protein/metabolism , Escherichia coli/metabolism , Histone Acetyltransferases/chemistry , Histone Acetyltransferases/metabolism , Ligands , Nuclear Receptor Coactivator 1 , Protein Binding , Receptors, Glucocorticoid/chemistry , Receptors, Glucocorticoid/metabolism , Recombinant Proteins , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid-myeloid lineage. Its biological role remains unknown. Using microarrays, we identified CD53 as one of the principal genes up-regulated by exposure of macrophages to LPS. Northern blot analysis confirmed the induction of CD53 in RAW264.7 macrophages treated with LPS or SNAP (a nitric oxide donor). Cells stably transfected with sense CD53 cDNA had increased levels of intracellular GSH and lower levels of peroxide, and were more resistant to H2O2 and to UVB irradiation. Cells harboring antisense CD53 had the opposite properties. We propose that the induction of CD53 is a major mechanism by which macrophages protect themselves against LPS-induced oxidative stress and UVB irradiation.
Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Lipopolysaccharides/metabolism , Macrophages/drug effects , Macrophages/radiation effects , Oxidative Stress , Penicillamine/analogs & derivatives , Animals , Biological Transport , Blotting, Northern , Blotting, Western , Cell Line , Cell Survival , DNA, Complementary/metabolism , Flow Cytometry , Glutathione/metabolism , Hydrogen Peroxide/pharmacology , Macrophages/metabolism , Mice , Nitric Oxide Donors/pharmacology , Oligonucleotide Array Sequence Analysis , Penicillamine/pharmacology , Polymerase Chain Reaction , RNA/metabolism , Reactive Oxygen Species , Reverse Transcriptase Polymerase Chain Reaction , Tetraspanin 25 , Time Factors , Transfection , Ultraviolet Rays , Up-Regulation , gamma-Glutamyltransferase/metabolismABSTRACT
Sauchinone, a known lignan, was isolated from the root of Saururus chinensis as an active principle responsible for inhibiting the production of NO in LPS-stimulated RAW264.7 cells by activity-guided fractionation. Sauchinone dose-dependently inhibited not only the production of NO, but also the expression of iNOS mRNA and protein in LPS-stimulated RAW 264.7 cells. Furthermore, sauchinone prevented LPS-induced NF-kappaB activation, which is known to play a critical role in iNOS expression, assessed by a reporter assay under the control of NF-kappaB. However, an electrophoretic mobility shift assay (EMSA) demonstrated that sauchinone did not suppress the DNA-binding activity of NF-kappaB or the degradation of IkappaB-alpha induced by LPS. Further analysis revealed that transactivation activity of RelA subunit of NF-kappaB was dose-dependently suppressed in the presence of sauchinone. Taken together, our results suggested that sauchinone could inhibit production of NO in LPS-stimulated RAW264.7 cells through the suppression of NF-kappaB by inhibiting transactivation activity of RelA subunit.
Subject(s)
Benzopyrans/pharmacology , Dioxoles/pharmacology , Nitric Oxide Synthase/drug effects , Phytotherapy , Saururaceae , Animals , Benzopyrans/administration & dosage , Benzopyrans/therapeutic use , Blotting, Western , Dioxoles/administration & dosage , Dioxoles/therapeutic use , Dose-Response Relationship, Drug , Electrophoretic Mobility Shift Assay , Gene Expression Regulation, Enzymologic , HeLa Cells/drug effects , HeLa Cells/metabolism , Humans , Lignans/administration & dosage , Lignans/pharmacology , Lignans/therapeutic use , Lipopolysaccharides , Macrophages/drug effects , Macrophages/metabolism , Mice , NF-kappa B/metabolism , Nitric Oxide Synthase/metabolism , Plant Extracts/administration & dosage , Plant Extracts/pharmacology , Plant Extracts/therapeutic use , Plant Roots , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The inducer of differentiation of human promyelocytic leukemia HL-60 cells is commonly accepted to have potential therapeutic importance. Verticinone, one of the major isosteroidal alkaloids from the bulbus of Fritillaria ussuriensis, was found to inhibit the growth of HL-60 cells by inducing these cells to differentiate toward granulocytes. Importantly, the combination of verticinone with all-trans retinoic acid (ATRA), a well-known inducer of HL-60 cells into granulocytic lineages, was more effective than either alone, suggesting its therapeutic use in minimizing the effective dose of ATRA.