Regulation of Nramp1 mRNA stability by oxidants and protein kinase C in RAW264.7 macrophages expressing Nramp1(Gly169)

The murine Nramp1 (natural-resistance-associated macrophage protein) locus confers innate resistance against intracellular macrophage pathogens. The g ene encodes a transporter molecule, which is rapidly recruited to the phago some. Nramp1 Functions as an iron transporter by transporting iron into the phagosome. Within the phagosome iron mediates anti-microbial killing by hy droxyl radical formation through the iron-catalysed Fenton/Haber-Weiss reac tion. In addition to its effects on the growth of intracellular pathogens, Nramp1 exerts a wide range of pleiotropic effects in activated macrophages. One of these pleiotropic effects is the increased stability of a variety o f mRNA species, including Nramp1 mRNA. In the present study, the stability of Nramp1 mRNA in Mycobacterium avium infected RAW264.7 macrophages express ing either the Nramp1(Gly169) resistant allele or the Nramp1(Asp169) suscep tible allele was examined, Nramp1 mRNA stability was greater in Nf Nramp1(G ly169) macrophages than in Nramp1(Asp169) macrophages. The increase in Nram p1 mRNA stability in resistant macrophages was inhibited by antioxidants an d protein kinase C (PKC) inhibitors, suggesting that Nramp1 mRNA stability is regulated by an oxidant-generated signalling pathway that requires PKC a ctivity. This was corroborated by treating Nramp1(Asp169) macrophages with menadione, which generates reactive oxygen species within cells. Menadione increased Nramp1 mRNA stability to the level observed in resistant macropha ges; this increase was also inhibited by a PKC inhibitor. Further, PKC acti vity was found to be greater in M. avium-infected Nramp1(Gly169) macrophage s than in infected Nramp1(Asp169) macrophages and inhibited by treatment wi th an antioxidant.