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

Abstract

The murine Nramp1 (natural-resistance-associated macrophage protein) locus confers innate resistance against intracellular macrophage pathogens. The gene encodes a transporter molecule, which is rapidly recruited to the phagosome. Nramp1 functions as an iron transporter by transporting iron into the phagosome. Within the phagosome iron mediates anti-microbial killing by hydroxyl radical formation through the iron-catalysed Fenton/Haber-Weiss reaction. 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 of mRNA species, including Nramp1 mRNA. In the present study, the stability of Nramp1 mRNA in Mycobacterium avium infected RAW264. 7 macrophages expressing either the Nramp1(Gly169) resistant allele or the Nramp1(Asp169) susceptible allele was examined. Nramp1 mRNA stability was greater in Nramp1(Gly169) macrophages than in Nramp1(Asp169) macrophages. The increase in Nramp1 mRNA stability in resistant macrophages was inhibited by antioxidants and protein kinase C (PKC) inhibitors, suggesting that Nramp1 mRNA stability is regulated by an oxidant-generated signalling pathway that requires PKC activity. 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 macrophages; this increase was also inhibited by a PKC inhibitor. Further, PKC activity was found to be greater in M. avium-infected Nramp1(Gly169) macrophages than in infected Nramp1(Asp169) macrophages and inhibited by treatment with an antioxidant.