Dual activity of pyrrolidine dithiocarbamate on kappaB-dependent gene expression in U937 cells: II. Regulation by tumour necrosis factor-alpha.
In the human promonocytic U937 cell line, pyrrolidine dithiocarbamate (PDTC) was a potent inhibitor of the nuclear factor-kappaB (NF-kappaB) signalling pathway induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). However, PDTC did not inhibit tumour necrosis factor-alpha (TNF-alpha)-induced NF-kappaB DNA binding activity but potentiated the effect of TNF-alpha on kappaB-dependent gene expression. The stimulatory effect of PDTC with TNF-alpha was not observed with an HIV-1 LTR reporter construct containing two mutated kappaB binding sites or with a construct with a mutation of the activating protein (AP)-2 binding site located between the two kappaB elements. Two distinct signalling pathways, one mediated by TPA and the other by TNF-alpha, were shown to interact, functionally defining a threshold important in the inhibitory or stimulatory effect of PDTC on kappaB-dependent gene expression. Evidence that PDTC induced AP-1 DNA binding and AP-1 reporter gene activity, raised the hypothesis that the effect of PDTC was mediated by an interaction between the AP-1 pathway and p65(RelA). Co-transfection with expression vectors for p65(RelA) and the AP-1 subunits c-Fos and c-Jun resulted in a decrease in the stimulatory effect of PDTC on HIV-1 LTR activity. Co-transfection of p65(RelA) with Tam67, a dominant negative mutant of c-Jun defective in transactivation, stimulated the effect of PDTC on HIV-1 LTR activity. Evidence that the stimulatory effect of Tam67 with PDTC was reduced with c-Jun is consistent with the hypothesis.
Watanabe
K
K
Signal Transduction Laboratory, Pharmaceutical Discovery Division, SRI International, Menlo Park, CA 94025, USA.
Kazakova
I
I
Furniss
M
M
Miller
S C
SC
D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D016325:HIV Long Terminal Repeat; D006801:Humans; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D011759:Pyrrolidines / Q000378:metabolism* / Q000494:pharmacology; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013859:Thiocarbamates / Q000378:metabolism* / Q000494:pharmacology; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000378:metabolism; D050656:Transcription Factor AP-2; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D020298:U937 Cells
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011759:Pyrrolidines; D013859:Thiocarbamates; D018808:Transcription Factor AP-1; D050656:Transcription Factor AP-2; D051996:Transcription Factor RelA; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C020972:pyrrolidine dithiocarbamic acid; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D013755:Tetradecanoylphorbol Acetate