title: Pertussis toxin-sensitive heterotrimeric G(αi/o) proteins mediate WNT/β-catenin and WNT/ERK1/2 signaling in mouse primary microglia stimulated with purified WNT-3A.
abstract: 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/β-catenin signaling to regulate TCF/LEF-dependent gene expression. We have previously shown that β-catenin levels are elevated in proinflammatory microglia of Alzheimer's disease patients and that WNT-3A can evoke a strong proinflammatory response in primary microglia. In order to investigate the underlying mechanisms, we focus here on the pharmacological dissection of WNT-3A-induced signaling to β-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 Gαi/o proteins by pertussis toxin blocks WNT-3A-induced LRP6 phosphorylation, disheveled shift, β-catenin stabilization and phosphorylation of ERK1/2. On the other hand LRP6 blockade by Dickkopf 1 treatment abrogated the WNT/β-catenin pathway without affecting WNT/ERK1/2 signaling. In the opposite way, inhibition of βγ subunits, phospholipase C (PLC), intracellular calcium and MEK1/2, the upstream kinase of ERK1/2, blocked ERK1/2 phosphorylation but not β-catenin stabilization. In summary, the data suggest a central role of Gαi/o for both β-catenin-dependent and -independent pathways. WNT-3A-induced ERK1/2 phosphorylation is mediated by βγ 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 β-catenin-independent signaling downstream of WNT-3A is of physiological importance for the proinflammatory regulation of microglia.
authors.affiliation: Dept. Physiology & Pharmacology, Sec Receptor Biology & Signaling, Karolinska Institutet, S-17177 Stockholm, Sweden.
authors.forename: Carina
authors.initials: C
authors.lastname: Halleskog
authors.forename: Gunnar
authors.initials: G
authors.lastname: Schulte
mesh_terms: D000607:Aminoacetonitrile / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D060488:Low Density Lipoprotein Receptor-Related Protein-6 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D037342:Pertussis Toxin / Q000633:toxicity*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D010738:Type C Phospholipases / Q000378:metabolism; D051153:Wnt Proteins / Q000378:metabolism; D060509:Wnt3A Protein / Q000302:isolation & purification / Q000494:pharmacology*; D051176:beta Catenin / Q000378:metabolism
chemical_list: C501127:Dkk1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D060488:Low Density Lipoprotein Receptor-Related Protein-6; C555476:Lrp6 protein, mouse; D011994:Recombinant Proteins; C424127:SL 327; D051153:Wnt Proteins; D060509:Wnt3A Protein; D051176:beta Catenin; D000607:Aminoacetonitrile; D051546:Cyclooxygenase 2; D037342:Pertussis Toxin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D010738:Type C Phospholipases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium