Macrophage Cell-Conditioned Medium Promotes Cholinergic Differentiation of Undifferentiated Progenitors and Synergizes with Nerve Growth Factor Action in the Developing Basal Forebrain

Abstract

Conditioned medium from stimulated microglia and from the monocyte/macrophage cell line (RAW 264.7; MC-CM) promotes the differentiation of cholinergic neurons from undifferentiated progenitors in the septal nuclei and adjacent basal forebrain (BF). We have studied the regulation of this process by measuring the activity of choline acetyltransferase (ChAT) in cultured BF taken from embryonic day 16 rat brain. Inhibition of either xanthine oxidase with allopurinol or nitric oxide synthase with NG-monomethyl-l-arginine produces a small but significant improvement in the efficacy of MC-CM while inclusion of pyrrolidine dithiocarbamate, a hydroxyl radical scavenger widely used as an antioxidant, lowers MC-CM-induced ChAT activity. Addition of nerve growth factor (NGF) but not brain-derived neurotrophic factor or glial-derived neurotrophic factor together with MC-CM has a synergistic effect on both ChAT activity and ChAT mRNA, raising ChAT activity as much as 29-fold and ChAT mRNA almost 15-fold. While MC-CM raised mRNA for trkA, the effect was not synergistic with NGF. mRNA for the common neurotrophin receptor (p75NTR) showed a modest synergistic increase. Blockade of the Ras/Raf/ERK [extracellular signal-regulated kinase, also known as mitogen-activated protein [(MAP) kinase] signal transduction pathway with either PD28059 (an inhibitor of MAP kinase/ERK kinase kinase or MEK) or N-acetyl-S-farnesyl-l-cysteine (an inhibitor of Ras farnesylation and, hence, activation) inhibited the action of MC-CM. Moreover, a subpopulation of cells responded rapidly to MC-CM with an increased appearance of phosphorylated ERK. Because NGF also utilizes this pathway, synergy may occur along this signal transduction pathway.