Effects of protease inhibitors on LPS-mediated activation of a mouse macrophage cell line (J774)

Abstract

Pretreatment (1 h) of a mouse macrophage-like cell line, J774, with the protease inhibitor, phenylalanine-chloromethyl ketone (PCK) or its structural analogue, tosylphenylalanine chloromethyl ketone (TPCK) was found to cause substantial inhibition of LPS-triggered activation of NF-κB. Pretreatment of cells with other types of protease inhibitors or their various structural analogues had no effect. PCK or TPCK appeared to exert its inhibitory effect by: (i) partially preventing LPS-triggered degradation of IκBα protein; (ii) preventing LPS-triggered nuclear translocation of NF-κB proteins (p50, RelA and Rel); and (iii) inhibiting the DNA-binding activities of NF-κB proteins. Pretreatment of cells with PCK or TPCK also resulted in the total or partial inhibition of LPS activatable (AP-1 or CREB) or constitutively-existing (Oct-1) transcription factors, but not of another constitutively-expressed transcription factor (SP-1). Pretreatment of J774 cells with PCK was found to substantially suppress LPS-induced expression of mRNAs specific for cytokine genes (TNFα, IL-1α and β, and IL-6), inducible nitric oxide synthase (iNOS) gene and IκBα gene, but not NF-κB1 p105 gene or β-actin gene. Furthermore, PCK pretreatment inhibited, in a dose-dependent manner, LPS-triggered production of nitric oxide production and tumoricidal activity.