Cell Adhesion Signaling Regulates RANK Expression in Osteoclast Precursors

Ayako Mochizuki,Sakae Tanaka,Tsuyoshi Nakamaki,Shigeru Tomoyasu,Yuho Kadono,Ryutaro Kamijo,Tomio Inoue,Yoichi Miyamoto,Masamichi Takami,Serge Nataf

doi:10.1371/journal.pone.0048795

Ayako Mochizuki, Sakae Tanaka + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0048795

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Journal: PLoS ONE	Publication Date: Nov 6, 2012
Citations: 61	License type: CC BY 4.0

Affiliation: The University of Tokyo, Showa University

Abstract

Cells with monocyte/macrophage lineage expressing receptor activator of NF-κB (RANK) differentiate into osteoclasts following stimulation with the RANK ligand (RANKL). Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition) or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition). BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS) and tumor necrosis factor –αTNF-α activated the NF-κB-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6) in BMMs induced their differentiation into osteoclasts even under the non-adherent condition. These results suggest that cell adhesion signaling regulates RANK expression in osteoclast precursors.

Highlights

Osteoclasts, multinucleated giant cells that resorb bone, differentiate from hematopoietic cells with a monocyte/macrophage lineage [1,2,3]
Mature osteoclasts that differentiated under the adherent condition did not incorporate the zymosan particles (Fig. 2C), while bone marrow-derived macrophages (BMMs) that failed to differentiate into osteoclasts incorporated those particles (Fig. 2C), suggesting that BMMs under the non-adherent condition retained the function of macrophages
BMMs stimulated with receptor activator of NF-kB ligand (RANKL) under the adherent condition showed upregulation of osteoclast marker genes, such as NFATc1, av and b3 integrins, and osteoclast associated receptor (OSCAR), whereas macrophage specific genes such as FccR III and lysozyme were downregulated in a timedependent manner (Fig. 2D, E)

Summary

Introduction

Osteoclasts, multinucleated giant cells that resorb bone, differentiate from hematopoietic cells with a monocyte/macrophage lineage [1,2,3]. Osteoblasts produce the osteoclast differentiation factor receptor activator of NF-kB ligand (RANKL) (identical to TRANCE, ODF, OPGL, TNFSF11, and CD254) in response to several bone resorbing factors, such as 1a,25-dihydroxyvitamin D3 [1a,25(OH)2D3] and parathyroid hormone (PTH) [5,6,7]. Binding of RANKL to its receptor RANK on osteoclast precursors results in recruitment of TNF receptor-associated factor (TRAF) family proteins such as TRAF6, which activate NF-kB and MAP kinases (MAPKs). Such signaling subsequently activates the transcription factors c-fos, PU., and NFATc1, all of which are required for osteoclast differentiation [8,9]. Disruption of RANKL or RANK results in osteopetrosis due to impaired osteoclast differentiation [11,12], indicating that the RANK-RANKL system is essential for regulation of osteoclast differentiation in vivo

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