Abstract
Inflammation and TNF-alpha signaling play a central role in most of the pathological conditions where cell transplantation could be applied. As shown by initial experiments, embryonic stem (ES) cells and ES-cell derived vascular cells express very low levels of TNF-alpha receptor I (TNFRp55) and thus do not induce cytokine expression in response to TNF-alpha stimulation. Transient transfection analysis of wild-type or deletion variants of the TNFRp55 gene promoter showed a strong activity for a 250-bp fragment in the upstream region of the gene. This activity was abolished by mutations targeting the Sp1/Sp3 or AP1 binding sites. Moreover, treatment with trichostatin A (TSA) led to a pronounced increase in TNFRp55 mRNA and promoter activity. Overexpression of Sp1 or c-fos further enhanced the TSA-induced luciferase activity, and this response was attenuated by Sp3 or c-jun coexpression. Additional experiments revealed that TSA did not affect the Sp1/Sp3 ratio but caused transcriptional activation of the c-fos gene. Thus, we provide the first evidence that ES and ES-cell-derived vascular cells lack cytokine expression in response to TNF-alpha stimulation due to low levels of c-fos and transcriptional activation of Sp1 that can be regulated by inhibition of histone deacetylase activity.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Blotting, Western
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Cell Line
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Cells, Cultured
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Cytokines / genetics*
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Embryonic Stem Cells / cytology
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Embryonic Stem Cells / drug effects
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Embryonic Stem Cells / metabolism*
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Endothelial Cells / cytology
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Endothelial Cells / drug effects
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Endothelial Cells / metabolism*
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Enzyme Inhibitors / pharmacology
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Gene Expression / drug effects
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Histone Deacetylase Inhibitors
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Histone Deacetylases / metabolism
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Hydroxamic Acids / pharmacology*
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Interleukin-6 / genetics
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Mice
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Mutation
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Myocytes, Smooth Muscle / drug effects
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Myocytes, Smooth Muscle / metabolism*
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Promoter Regions, Genetic / genetics
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Proto-Oncogene Proteins c-fos / genetics
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Proto-Oncogene Proteins c-fos / metabolism
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Proto-Oncogene Proteins c-jun / genetics
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RNA, Small Interfering / genetics
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Receptors, Tumor Necrosis Factor, Type I / genetics
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Receptors, Tumor Necrosis Factor, Type I / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Sp1 Transcription Factor / genetics
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Sp3 Transcription Factor / genetics
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Transfection
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Tumor Necrosis Factor-alpha / genetics
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Tumor Necrosis Factor-alpha / pharmacology*
Substances
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Cytokines
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Enzyme Inhibitors
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Histone Deacetylase Inhibitors
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Hydroxamic Acids
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Interleukin-6
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Proto-Oncogene Proteins c-fos
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Proto-Oncogene Proteins c-jun
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RNA, Small Interfering
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Receptors, Tumor Necrosis Factor, Type I
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Sp1 Transcription Factor
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Tumor Necrosis Factor-alpha
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Sp3 Transcription Factor
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trichostatin A
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Histone Deacetylases