ABSTRACT
Studies in yeast demonstrate that signaling kinases have a surprisingly active role in the nucleus, where they tether to chromatin and modulate gene expression programs. Despite these seminal studies, the nuclear mechanism of how signaling kinases control transcription of mammalian genes is in its infancy. Here, we provide evidence for a hitherto unknown function of protein kinase C-theta (PKC-θ), which physically associates with the regulatory regions of inducible immune response genes in human T cells. Chromatin-anchored PKC-θ forms an active nuclear complex by interacting with RNA polymerase II, the histone kinase MSK-1, and the adaptor molecule 14-3-3ζ. ChIP-on-chip reveals that PKC-θ binds to promoters and transcribed regions of genes, as well as to microRNA promoters that are crucial for cytokine regulation. Our results provide a molecular explanation for the role of PKC-θ not only in normal T cell function, but also in circumstances of its ectopic expression in cancer.
Subject(s)
Chromatin/metabolism , Gene Expression Regulation , Isoenzymes/metabolism , MicroRNAs/metabolism , Protein Kinase C/metabolism , T-Lymphocytes/physiology , Cell Line, Tumor , Cell Nucleus/metabolism , Humans , Interleukin-2/genetics , Isoenzymes/genetics , Jurkat Cells , MicroRNAs/genetics , Promoter Regions, Genetic , Protein Kinase C/genetics , Protein Kinase C-theta , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , T-Lymphocytes/cytology , Transcription, GeneticABSTRACT
Orlistat, an antiobesity drug, is cytostatic and cytotoxic to tumor cells. The antitumor activity of orlistat can be attributed to its ability to inhibit the thioesterase domain of fatty acid synthase (FAS). The objective of the present study was to test the effect of orlistat on endothelial cell proliferation and angiogenesis. Orlistat inhibits endothelial cell FAS, blocks the synthesis of fatty acids, and prevents endothelial cell proliferation. More significantly, orlistat inhibits human neovascularization in an ex vivo assay, which suggests that it may be useful as an antiangiogenic drug. The mechanism of these effects can be traced to the fact that orlistat prevents the display of the vascular endothelial growth factor (VEGF) receptor (VEGFR2/KDR/Flk1) on the endothelial cell surface. Thus, orlistat is an antiangiogenic agent with a novel mechanism of action.
Subject(s)
Cell Proliferation/drug effects , Endothelial Cells/cytology , Endothelium, Vascular/cytology , Lactones/pharmacology , Neovascularization, Pathologic/prevention & control , Neovascularization, Physiologic/drug effects , Cells, Cultured , Endothelial Cells/drug effects , Fatty Acid Synthases/antagonists & inhibitors , Humans , Neovascularization, Pathologic/drug therapy , Orlistat , Receptors, Vascular Endothelial Growth Factor/analysis , Receptors, Vascular Endothelial Growth Factor/drug effects , Umbilical Veins/cytologyABSTRACT
OBJECTIVE: Although heparanase is recognized as a proangiogenic factor, the involvement of heparanase in rheumatoid arthritis (RA) is unclear. In this study, we assessed heparanase activity in synovial fluid (SF) and synovial tissue (ST) from patients with RA or osteoarthritis (OA), and analyzed the expression of angiogenic pathway-focused genes in ST from RA and OA patients. METHODS: SF and ST were obtained from the knees of patients with either RA or OA and from asymptomatic donors with no documented history of degenerative or inflammatory joint diseases. Heparanase activity was determined by an enzymatic assay using a radiolabeled substrate, and the presence of heparanase in ST was demonstrated by Western blotting. The expression of angiogenesis genes, including heparanase, in ST was analyzed by real-time quantitative polymerase chain reaction. RESULTS: Heparanase activity was dramatically higher (>100-fold) in SF and ST from RA patients than in SF and ST from OA patients and asymptomatic donors. Active heparanase enzyme was detected and heparanase messenger RNA was up-regulated in ST from RA patients. We also found that angiogenesis gene expression was significantly regulated in RA synovium, and was correlated with heparanase activity. CONCLUSION: These findings are novel and contribute to our understanding of joint destruction in RA, suggesting that heparanase may be a reliable prognostic factor for RA progression and an attractive target for the treatment of RA.