Antisense oligonucleotides targeting protein kinase C-alpha, -beta I, or -delta but not -eta inhibit lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages: involvement of a nuclear factor kappa B-dependent mechanism

Abstract

The signaling pathway for protein kinase C (PKC) activation and the role of PKC isoforms in LPS-induced nitric oxide (NO) release were studied in RAW 264.7 macrophages. The tyrosine kinase inhibitor genestein attenuated LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression, as did the phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122 and the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609. LPS stimulated phosphatidylinositol (PI) hydrolysis and PKC activity in RAW cells; both were inhibited by genestein. The PKC inhibitors (staurosporine, calphostin C, Ro 31-8220, or Go 6976) or long-term 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment also resulted in inhibition of LPS-induced NO release and iNOS expression. Western blot analysis showed expression of PKC-alpha, -betaI, -delta, -eta, and -zeta in RAW cells; down-regulation of PKC-alpha, -betaI, and -delta, but not -eta, was seen after long-term TPA treatment, indicating the possible involvement of one or all of PKC-alpha, -betaI, and -delta, but not -eta, in LPS-mediated effects. Treatment with antisense oligonucleotides for these isoforms further demonstrated the involvement of PKC-alpha, -betaI, and delta, but not -eta, in LPS responses. Stimulation of cells with LPS for 1 h caused activation of NF-kappaB in the nuclei by detection of NF-kappaB-specific DNA-protein binding; this was inhibited by genestein, U73122, D609, calphostin C, or antisense oligonucleotides for PKC-alpha, -betaI, and -delta, but not -eta. These data suggest that LPS activates PI-PLC and PC-PLC via an upstream tyrosine kinase to induce PKC activation, resulting in the stimulation of NF-kappaB DNA-protein binding, then initiated the expression of iNOS and NO release. PKC isoforms alpha, betaI, and delta were shown to be involved in the regulation of these LPS-induced events.