Regulation of the RON receptor tyrosine kinase expression in macrophages: Blocking the RON gene transcription by endotoxin-induced nitric oxide

Previous studies have shown that activation of the RON receptor tyrosine ki nase inhibits inducible NO production in murine peritoneal macrophages, The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-gamma, and TNF-alpha regulate RON expression. Western blot anal ysis showed that RON expression is reduced in peritoneal macrophages collec ted from mice injected with a low dose of LPS, The inhibition was seen as e arly as 8 h after LPS challenge, Experiments in vitro also demonstrated tha t the levels of the RON mRNA and protein are diminished in cultured periton eal macrophages following LPS stimulation. TNF-alpha plus IFN-gamma abrogat ed macrophage RON expression, although individual cytokines had no signific ant effect. Because LPS and TNF-alpha plus IFN-gamma induce NO production, we reasoned that NO might be involved in the RON inhibition, Two NO donors, S-nitroglutathione (GSNO) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) , directly inhibited macrophage RON expression when added to the cell cultu res, Blocking NO production by NO inhibitors like TGF-beta prevented the LP S-mediated inhibitory effect. In Raw264.7 cells transiently transfected wit h a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-s timulating protein-induced RON phosphorylation and macrophage migration, We concluded from these data that RON expression in macrophages is regulated during inflammation, LPS and TNF-alpha plus IFN-gamma are capable of down-r egulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activitie s.