Macrophage colony-stimulating factor (CSF-1) binds to a receptor (CSF-1R) encoded by the c- fms proto-oncogene and activates transcription of the urokinase plasminogen activator (uPA) gene in murine bone-marrow-derived macrophages. This article demonstrates that the murine macrophage cell line RAW264 responds to CSF-1 with inducible phosphorylation of cytoplasmic proteins on tyrosine residues but fails to induce transcription of uPA. The defect was correlated with a selective failure to maintain CSF-1Rs on the cell surface, whereas all RAW264 cells contained abundant CSF-1Rs within the presumptive Golgi/endoplasmic reticulum compartment. Transfection with a CSF-1R expression plasmid permitted CSF-1-dependent activation of the signalling pathway targeting an Ets/AP1 (activator protein 1) element in the uPA promoter that has been shown previously to be a target of oncogenic ras and protein kinase C pathways. Mutation of the expressed CSF-1R at either Y807 or Y559, sites of receptor tyrosine phosphorylation implicated in signal transduction, reduced but did not abolish uPA promoter activation by CSF-1. Activation by mutant CSF-1R plasmids was additive; there was no evidence of mutual complementation. The results indicate that maintenance of elevated uPA transcription by CSF-1 requires new receptors emerging continuously on the cell surface. Parallel, partly redundant, signalling pathways arising from phosphorylated tyrosines on the CSF-1R activate multiple cis -acting elements on the complex uPA promoter.
Activated macrophages require L -arginine uptake to sustain NO synthesis. Several transport systems could mediate this L -arginine influx. Using competition analysis and gene-expression studies, amino acid transport system y + was identified as the major carrier responsible for this activity. To identify which of the four known y + transport-system genes is involved in macrophage-induced L -arginine uptake, we used a hybrid-depletion study in Xenopus oocytes. Cationic amino acid transporter (CAT) 2 antisense oligodeoxyribonucleotides abolished the activated-macrophage-mRNA-induced L -arginine transport. Together with expression studies documenting that CAT2 mRNA and protein levels are elevated with increased L -arginine uptake, our data demonstrate that CAT2 mediates the L -arginine transport that is required for the raised NO production in activated J774 macrophages.
1. The role of enhanced aerobic glycolysis in the transformation of rat thymocytes by concanavalin A has been investigated. Concanavalin A addition doubled [U- 14 C]glucose uptake by rat thymocytes over 3h and caused an equivalent increased incorporation into protein, lipids and RNA. A disproportionately large percentage of the extra glucose taken up was converted into lactate, but concanavalin A also caused a specific increase in pyruvate oxidation, leading to an increase in the percentage contribution of glucose to the respiratory fuel. 2. Acetoacetate metabolism, which was not affected by concanavalin A, strongly suppressed pyruvate oxidation in the presence of [U- 14 C]glucose, but did not prevent the concanavalin A-induced stimulation of this process. Glucose uptake was not affected by acetoacetate in the presence or absence of concanavalin A, but in each case acetoacetate increased the percentage of glucose uptake accounted for by lactate production. 3. [ 3 H]Thymidine incorporation into DNA in concanavalin A-treated thymocyte cultures was sensitive to the glucose concentration in the medium in a biphasic manner. Very low concentrations of glucose (25μ m ) stimulated DNA synthesis half-maximally, but maximum [ 3 H]thymidine incorporation was observed only when the glucose concentration was raised to 1m m . Lactate addition did not alter the sensitivity of [ 3 H]-thymidine uptake to glucose, but inosine blocked the effect of added glucose and strongly inhibited DNA synthesis. 4. It is suggested that the major function of enhanced aerobic glycolysis in transforming lymphocytes is to maintain higher steady-state amounts of glycolytic intermediates to act as precursors for macromolecule synthesis.