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 G_αi/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 G_αi/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.

Introduction

WNTs are secreted lipoglycoproteins, which bind and activate the Class Frizzled (FZD) surface receptors [1], [2]. In mammals 19 different WNTs are known and WNT-3A belongs to the WNT-1 group of ligands, which generally activate the WNT/β-catenin pathway [3], [4]. This signaling route involves establishment of a WNT-FZD-LRP5/6 ternary complex initiating the formation of disheveled (DVL)-dependent signalosomes [5]. Scaffolding of several signaling compounds, such as DVL and axin in turn mediates inhibition of the glycogen synthase kinase (GSK3)-dependent destruction complex of β-catenin. Thereby, constitutive phosphorylation of β-catenin by GSK3 and subsequent proteasomal degradation of β-catenin are blocked and β-catenin accumulates to act as a transcriptional regulator [5]. In addition to WNT/β-catenin signaling an ever growing network of β-catenin-independent signaling pathways emerges, where the phosphoprotein DVL and heterotrimeric G proteins appear at the crossroads of most FZD-mediated pathways [6], [7], [8]. Several studies have emphasized the importance of heterotrimeric G proteins for WNT/β-catenin signaling, but the relative localization and mechanistic contribution of the G protein in the WNT pathway remains elusive [9], [10], [11].

Microglia, the macrophages of the brain [12], contain an elaborate setup of WNT receptors enabling them to respond to both WNT-1- and WNT-5-like WNTs, acting through β-catenin-dependent and β-catenin-independent pathways, respectively [13], [14], [15], [16]. Elevated β-catenin levels in microglia of the brains from Alzheimer's disease and old patients indicate that WNT-induced β-catenin signaling is correlated with the activation status of this cell type in vivo [14]. In fact, WNT-3A activates the β-catenin dependent signaling pathway – monitored as β-catenin stabilization and nuclear translocation – in both mouse primary microglia and a microglia-like cell line called N13 and evokes a proinflammatory transformation [14]. Studies in N13 cells revealed also that WNT-3A is capable to activate heterotrimeric G proteins in addition to the WNT/β-catenin pathway [16]. Further, we reported that WNT-5A stimulation of mouse primary microglia reduced intracellular cAMP levels and induced phospholipase-dependent activation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinase1/2 (ERK1/2) in a G_αi/o-dependent manner [13]. This signaling pathway mediates several aspects of the WNT-5A-induced proinflammatory transformation, such as matrix metalloprotease 9/13 expression, invasion and proliferation [13]. MAPKs, such as the ERK1/2 are well-established regulators of microglia function [17]. In addition, this group of kinases has been assigned an important yet poorly defined role in the regulation of and crosstalk with the WNT/β-catenin pathway on different levels: modulation of LRP5/6, GSK3 and transcriptional regulation [18], [19], [20].

Here we show that WNT-3A induces ERK1/2 phosphorylation and activation of WNT/β-catenin signaling with different kinetics. Even though both pathways are activated in parallel pharmacological tools are clearly able to separate between pertussis toxin-sensitive LRP5/6-dependent stabilization of β-catenin and LRP5/6-independent, heterotrimeric Gβγ protein-mediated activation of ERK1/2 signaling. Thus, our study argues for a central role of heterotrimeric G_αi/o proteins in integrating WNT-3A signaling. We show that WNT-3A can activate both β-catenin-dependent and -independent signaling in mouse primary microglia cells with natural receptor stoichiometry. Furthermore, it becomes obvious that LRP5/6-independent signaling through heterotrimeric G proteins towards ERK1/2 is of physiological importance for proinflammatory transformation of microglia.