Dual Activity of Pyrrolidine Dithiocarbamate on κB-Dependent Gene Expression in U937 Cells: II. Regulation by Tumour Necrosis Factor-α

Abstract

In the human promonocytic U937 cell line, pyrrolidine dithiocarbamate (PDTC) was a potent inhibitor of the nuclear factor-κB (NF-κB) signalling pathway induced by the phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA). However, PDTC did not inhibit tumour necrosis factor-α (TNF-α)-induced NF-κB DNA binding activity but potentiated the effect of TNF-α on κB-dependent gene expression. The stimulatory effect of PDTC with TNF-α was not observed with an HIV-1 LTR reporter construct containing two mutated κB binding sites or with a construct with a mutation of the activating protein (AP)-2 binding site located between the two κB elements. Two distinct signalling pathways, one mediated by TPA and the other by TNF-α, were shown to interact, functionally defining a threshold important in the inhibitory or stimulatory effect of PDTC on κB-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.