Cell Adhesion Signaling Regulates RANK Expression in Osteoclast Precursors

<div><p>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.</p> </div

Schema of adherent and non-adherent cell culture systems used in this study.

<p>BMMs (1.25×10⁵/cm²) were cultured on plastic cell culture plates (<i>left</i>: adherent condition) or methylcellulose medium (<i>right</i>: non-adherent condition) in the presence of M-CSF (50 ng/ml), TGF-β (1.0 ng/ml), and RANKL (150 ng/ml) to examine osteoclast differentiation.</p

Osteoclast differentiation induced by forced expression of RANK and TRAF6 in BMMs under adherent and non-adherent condition.

<p>BMMs over-expressing RANK (transduced with pMX-RANK-puro vector) or TRAF6 (transduced with pMX-TRAF6-puro vector) were cultured with M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) for 4 days under the adherent or non-adherent condition, then fixed and stained for TRAP. Cell under non-adherent condition were harvested and placed on cell culture plates for 1 hour to stain for TRAP.</p

Osteoclast differentiation under adherent and non-adherent conditions.

<p><b><i>A</i></b><i>.</i> BMMs were cultured with M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) under the adherent and non-adherent conditions for 24, 48, 72, or 96 hours, after which non-adherent cells were harvested and placed on plastic cell culture plates for 30 minutes, then fixed and stained for TRAP. Cells shown stained red are TRAP-positive cells. <b><i>B</i></b><i>.</i> BMMs grown under adherent and non-adherent conditions were cultured in the presence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) for 0, 24, 48, 72, or 96 hours. Subsequently, TRAP activity was determined. Data represent mean values of three independent experiments, with error bars indicating ± SD. <b><i>C</i></b><i>.</i> FITC-conjugated zymosan particles were added to BMMs after 0 and 96 hours of culturing under the adherent and non-adherent conditions in the presence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml). One hour after zymosan addition, cells were washed with PBS and fixed, then the zymosan particles were visualized by UV illumination. Green dots indicate FITC-conjugated zymosan particles incorporated into cells. <b><i>D</i></b><i>.</i> BMMs were cultured in the presence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) under the adherent and non-adherent conditions for 0, 12, 24, 48, and 72 hours, after which mRNA expression levels were examined by RT-PCR. <b><i>E and F</i></b><i>.</i> Relative mRNA expression levels of NFATc1 (<b><i>E</i></b>) and integrin β₃ (<b><i>F</i></b>) at the indicated times under the adherent and non-adherent conditions in the presence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml), which were quantified using real-time RT-PCR. Data represent the mean values of three independent experiments, with error bars indicating ± SD. **<i>P</i>< 0.01 vs adherent condition at same time point.</p

Effects of RANKL, TNF-α, and LPS on activation of NF-κB pathway.

<p><b><i>A–C</i></b><i>.</i> Cell lysates of BMMs stimulated with RANKL (150 ng/ml) (<i>A</i>), TNF-α (10 ng/ml) (<i>B</i>), or LPS (1 µg/ml) (<b><i>C</i></b>) for the indicated time periods under the adherent and non-adherent conditions were harvested, then phosphorylation and subsequent degradation of IκB were examined by immunoblot analysis. <b><i>D</i></b><i>.</i> BMMs cultured under the adherent and non-adherent conditions were treated with LPS (1 µg/ml) for 3 hours, after which total RNA was extracted and TNF-α mRNA expression levels were examined by RT-PCR.</p

RANK expression levels under adherent and non-adherent conditions.

<p><b><i>A</i></b><i>.</i> BMMs under the adherent condition were harvested (0 h) and subsequently cultured again under adherent and non-adherent conditions in the presence or absence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) for the indicated periods. Relative expression levels of RANK mRNA were determined by real time RT-PCR. Data represent mean values of three independent experiments, with error bars indicating ± SD. **<i>P</i>< 0.01 for adherent condition vs. non-adherent condition at the same time point. <b><i>B</i></b><i>. Left panel</i>: BMMs grown under the adherent condition were transferred to the non-adherent condition and further cultured for the indicated time periods. <i>Right panel</i>: BMMs under the non-adherent condition were transferred to the adherent condition and further cultured for the indicated time periods. RANK mRNA expression was analyzed by RT-PCR. <b><i>C</i></b>. <i>Left panel</i>: comparison of RANK protein expression levels between BMMs cultured under the adherent condition and those transferred to the non-adherent condition. <i>Right panel</i>: Comparison of RANK protein levels between BMMs cultured under the non-adherent condition and those transferred to the adherent condition. RANK protein expression on the surface of BMMs was analyzed by flowcytometry.</p

Effects of echistatin on osteoclast differentiation and RANK expression.

<p><b><i>A</i></b><i>.</i> BMMs were cultured in the presence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) with or without echistatin (10^-8 M). After culturing for 96 hours, cells were fixed and stained for TRAP (<b><i>A</i></b>), then TRAP activities were measured (<b><i>B</i></b>) and RANK expression levels evaluated (<b><i>C</i></b>). Data represent mean values of three independent experiments, with error bars indicating + SD. **<i>P</i><0.01.</p