Tosylphenylalanine chloromethyl ketone inhibits TNF-α mRNA synthesis in the presence of activated NF-κB in RAW 264.7 macrophages

Abstract

Serine proteinase inhibitors such as N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK) were shown to inhibit production of tumour necrosis factor-α (TNF- α) in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. The proteinase inhibitors were also reported to inhibit activation of the transcription factor nuclear factor-κB (NF-κB) by blocking the signalling pathway for stimuli-induced phosphorylation of the inhibitory subunit (IκBα) and thus preventing its degradation. In RAW 264.7 cells TPCK and TLCK significantly suppressed LPS-induced increase in TNF-α mRNA, induction of nuclear κB-binding activity and degradation of IκBα. TPCK and TLCK effectively blocked TNF-α mRNA synthesis even when they were added after LPS stimulation. In these cells, however, the inhibitory modes of the two inhibitors were found to be different: while addition of TLCK suppressed IκBα degradation and reduced NF-κB activity, a comparable decrease in the nuclear κB-binding activity or in IκBα degradation was not observed in cells treated with TPCK. Our results show that TPCK inhibits LPS-induced TNF-α mRNA synthesis in the presence of activated NF- κB and suggests that mechanisms other than NF-κB activation are involved in the transcriptional regulation of the TNF-α gene.