Protease inhibitors block lipopolysaccharide induction of tissue factor gene expression in human monocytic cells by preventing activation of c-Rel/p65 heterodimers.
Tissue factor (TF) is expressed rapidly by human monocytes exposed to bacterial endotoxin (lipopolysaccharide, or LPS). Transcriptional regulation is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site in the TF promoter. Nuclear translocation of cytosolic c-Rel/p65 heterodimers and other members of the NF-kappa B/Rel family requires dissociation and proteolytic degradation of the inhibitor protein, I kappa B alpha. The protease inhibitors N alpha-tosylphenylalanyl chloromethyl ketone (TPCK) and N alpha-tosyl-L-lysine chloromethyl ketone (TLCK) block activation of NF-kappa B/Rel proteins by preventing degradation of I kappa B alpha. To determine if TPCK and TLCK inhibited LPS induction of TF expression, freshly isolated human monocytes and monocytic THP-1 cells were pretreated with these inhibitors for 30 min before LPS stimulation. Both TPCK and TLCK inhibited LPS induction of TF protein, TF mRNA and TF promoter activity in a dose-dependent manner. These inhibitors specifically prevented degradation of I kappa B alpha and nuclear translocation of c-Rel/p65 heterodimers. In contrast, TPCK and TLCK did not block induction of an immediate-early gene encoding the transcription factor, Egr-1. Taken together, these data indicated that inhibiting nuclear translocation of c-Rel/p65 heterodimers prevented LPS induction of TF gene transcription in monocytic cells.
Mackman
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Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
D001483:Base Sequence; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D051766:Early Growth Response Protein 1; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D017874:Immediate-Early Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D020569:Proto-Oncogene Proteins c-rel; D012333:RNA, Messenger / Q000032:analysis; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D014157:Transcription Factors / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000187:drug effects
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D008070:Lipopolysaccharides; D011480:Protease Inhibitors; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D012333:RNA, Messenger; D014157:Transcription Factors; D014107:Tosyllysine Chloromethyl Ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D013925:Thromboplastin