Long-Lasting Activity of ERK Kinase Depends on NFATc1 Induction and Is Involved in Cell Migration-Fusion in Murine Macrophages RAW264.7.

Roberta Russo,Selene Mallia,Nadia Lampiasi,Francesca Zito

doi:10.3390/ijms21238965

Abstract

Macrophages are mononuclear cells that become osteoclasts (OCs) in the presence of two cytokines, macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL). RANKL binding to its specific receptor RANK leads to OCs differentiation mainly by nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). In our previous study, the analysis of the protein network in NFATc1-knockdown cells, using the Ingenuity Pathway Analysis (IPA), showed a link between NFATc1 and Mitogen-activated protein kinase kinase (MEK)-extracellular receptor kinase (ERK) signaling pathway. Therefore, this study aimed to extend our knowledge of the relationship between NFATc1 and the ERK. Here, we demonstrate that delayed ERK1/2 phosphorylation in pre-OC RANKL-induced depends on NFATc1. Indeed, the knockdown of NFATc1 reduced the phosphorylation of ERK1/2 (60%) and the pharmacological inhibition of the ERK1/2 kinase activity impairs the expression of NFATc1 without preventing its translocation into the nucleus. Furthermore, silencing of NFATc1 significantly reduced RANKL-induced migration (p < 0.01), and most pre-OCs are still mononuclear after 48 h (80 ± 5%), despite the presence of actin rings. On the other hand, the inhibitors FR180204 and PD98059 significantly reduced RANKL-induced cell migration (p < 0.01), leading to a reduction in the number of multinucleated cells. Finally, we suggest that long-lasting ERK activity depends on NFATc1 induction and is likely linked to cell migration, fusion, and OC differentiation.

Highlights

OCs are multinucleated giant cells formed from hematopoietic progenitors in the monocyte/macrophage lineage [1]
We performed the analysis after 24 h of RANKL-induction, as it is the best time for the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1)-increase/extracellular receptor kinase (ERK)-phosphorylation and the beginning of pre-OCs differentiation
Lee and colleagues reported that RANKL induced an early (5 to 20 min) and a delayed (8 to 24 h) activation of p38 and extracellular receptor kinase 1/2 (ERK1/2) kinases in Bone marrow macrophages (BMMs) positively related with the onset of OCs differentiation [16]

Summary

Introduction

OCs are multinucleated giant cells formed from hematopoietic progenitors in the monocyte/macrophage lineage [1]. RANKL-RANK binding leads to RANK aggregates in trimers and this association transduces the intracellular signal through the recruitment of adapter proteins, including TNF receptor-associated factor 6 (TRAF6) [6,7]. TRAF6 is a crucial signaling protein that functions for different pathways, from adaptative and innate immunity to bone metabolism [8]. TRAF6 stimulates a cascade of transcription factors, including activator protein-1 (AP-1), c-Fos, microphthalmia-associated transcription factor (Mitf), PU., nuclear factor of activated T-cells cytoplasmic 2 (NFATc2) [9,10]. Among all the transcription factors already present in pre-OCs, NFATc1 is the only one strongly up-regulated and necessary-sufficient to induce differentiation of pre-OCs into OCs following RANKL-stimulation [11,12]

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