Pertussis toxin-sensitive heterotrimeric Gαi/o proteins mediate WNT/β-catenin and WNT/ERK1/2 signaling in mouse primary microglia stimulated with purified WNT-3A

WNT-3A is a secreted lipoglycoprotein that engages Class Frizzled receptors and LDL receptor related protein 5/6 (LRP5/6) for cellular communication. Generally, WNT-3A mediates WNT/beta-catenin signaling to regulate TCF/LEF-dependent gene expression. We have previously shown that beta-catenin levels are elevated in proinflammatory microglia of Alzheimer's disease patients and that WNT-3A can evoke a strong proinflammatoty response in primary microglia. In order to investigate the underlying mechanisms, we focus here on the pharmacological dissection of WNT-3A-induced signaling to beta-catenin and to the extracellular signal-regulated kinases 1/2 (ERK1/2) in mouse primary microglia. Both pathways are induced by WNT-3A with slightly different kinetics, suggesting that they might be pharmacologically separable. Inhibition of heterotrimeric G(alpha i/o) proteins by pertussis toxin blocks WNT-3A-induced LRP6 phosphotylation, disheveled shift, beta-catenin stabilization and phosphorylation of ERK1/2. On the other hand LRP6 blockade by Dickkopf I treatment abrogated the WNT/beta-catenin pathway without affecting WNT/ERK1/2 signaling. In the opposite way, inhibition of beta gamma subunits, phospholipase C (PLC), intracellular calcium and MEK1/2, the upstream kinase of ERK1/2, blocked ERK1/2 phosphotylation but not beta-catenin stabilization. In summary, the data suggest a central role of G(alpha i/o) both beta-catenin-dependent and -independent pathways. WVNT-3A-induced ERK1/2 phosphorylation is mediated by fly subunits, PLC, intracellular calcium and MEK1/2. Furthermore, we show that cyclooxygenase 2 (COX2), a generic proinflammatory marker of microglia, is induced by WNT-3A through ERK1/2-dependent pathways arguing that beta-catenin-independent signaling downstream of WNT-3A is of physiological importance for the proinflammatoty regulation of microglia. (C) 2012 Elsevier Inc. All rights reserved.