E-cadherin is important for cell differentiation during osteoclastogenesis

Bone 86 (2016) 106–118

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E-cadherin is important for cell differentiation during osteoclastogenesis Cara Fiorino, Rene E. Harrison ⁎ a b

Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario M1C 1A4, Canada Department of Biological Sciences, University of Toronto Scarborough, Toronto, Ontario M1C 1A4, Canada

a r t i c l e

i n f o

Article history: Received 3 November 2015 Revised 29 January 2016 Accepted 4 March 2016 Available online 6 March 2016 Keywords: E-cadherin Osteoclast Differentiation Cell–cell interaction NFATc1

a b s t r a c t E-cadherin, a protein responsible for intercellular adhesion between epithelial cells, is also expressed in the monocyte/macrophage lineage. In this study we have explored the involvement of E-cadherin during receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclast differentiation. Osteoclastogenesis involves a period of precursor expansion followed by multiple fusion events to generate a multinuclear osteoclast that is capable of bone resorption. We asked whether E-cadherin participated in early precursor interactions and recognition or was a component of the osteoclast fusion machinery. Here, we show that endogenous Ecadherin expression is the highest during early stages of osteoclast differentiation, with surface expression visible on small precursor cells (fewer than four nuclei per cell) in both RAW 264.7 cells and primary macrophages. Blocking E-cadherin function with neutralizing antibodies prior to the onset of fusion delayed the expression of TRAP, Cathepsin K, DC-STAMP and NFATc1 and significantly diminished multinucleated osteoclast formation. Conversely, E-cadherin-GFP overexpressing macrophages displayed earlier NFATc1 nuclear translocation along with faster formation of multinucleated osteoclasts compared to control macrophages. Through live imaging we identified that disrupting E-cadherin function prolonged the proliferative phase of the precursor population while concomitantly decreasing the proportion of migrating precursors. The lamellipodium and polarized membrane extensions appeared to be the principal sites of fusion, indicating precursor migration was a critical factor contributing to osteoclast fusion. These findings demonstrate that E-cadherin-mediated cell–cell contacts can modulate osteoclast-specific gene expression and prompt differentiating osteoclast precursors toward migratory and fusion activities. © 2016 Elsevier Inc. All rights reserved.

1. Introduction The health of a skeleton depends on a continuous and dynamic equilibrium between bone destruction and bone formation [1–4]. Osteoclasts are highly specialized multinucleated cells that perform a unique role in the bone remodeling process; they are singularly responsible for the selective resorption of bone matrix components [3,5]. Osteoclast-driven resorption is followed by matrix deposition by osteoblasts. This cooperative osteoclast–osteoblast relationship ensures the continuous remodeling of bone [2,4,6].

Abbreviations: DC-STAMP, dendritic cell-specific transmembrane protein; bAb, blocking antibody; pre-OC, pre-osteoclast; ICAM-2, intercellular adhesion molecule-2; Mac-1, macrophage antigen 1; ASCT2, alanine–serine–cysteine-preferring neutral amino acid transporter 2; SIRPα, signaling regulatory protein α; IL-4, interleukin-4; FBGC, foreign body giant cell; NFATc1, nuclear factor of activated T-cells, cytoplasmic 1; TRAP, tartrate resistant acid phosphatase; DIC microscopy, differential interference contrast microscopy; MMP-9, matrix metalloproteinase 9. ⁎ Corresponding author at: Department of Biological Sciences, University of Toronto Scarborough, Toronto, Ontario M1C 1A4, Canada. E-mail address: [email protected] (R.E. Harrison).

http://dx.doi.org/10.1016/j.bone.2016.03.004 8756-3282/© 2016 Elsevier Inc. All rights reserved.

Monocyte/macrophage precursor cells, derived from hematopoietic stem cells, receive recruitment signals (hormones, cytokines, etc.) to a target bone surface and interact with bone lining stromal/osteoblast cells, which produce M-CSF (macrophage colony stimulating factor) and express RANKL (receptor activator of nuclear factor-κB ligand) [4]. M-CSF and RANKL are critical for the regulation of osteoclastogenesis [5,7] and the discovery of these molecules has strongly contributed to the study of bone cell biology by providing a means to induce osteoclast formation without the need for co-culturing systems during in vitro analysis [4]. Osteoclast differentiation and activation is a highly regulated process. Commitment to the development of a mature, multinucleated osteoclast depends on multiple stages of differentiation. Osteoclastogenesis involves a period of precursor population expansion, followed by cell cycle withdrawal [8–10]. Cell cycle withdrawal prior to terminal differentiation has been documented in many cell types [11,12]. After cell cycle exit, in vitro osteoclastogenesis is marked by pre-OC migration, cell–cell interaction and eventually fusion. Cell–cell fusion only occurs naturally for a small number of cell types (myoblasts, trophoblasts, macrophages and gametes) in the mammalian system and is critical for the specialization of these cells [13,14]. It is believed that osteoclast

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resorption efficiency is largely determined by cell size, which is increased with each fusion event [4,15]. Cell–cell recognition and adhesion occur prior to membrane fusion of osteoclast precursor cells. A number of receptor/ligand interactions including ICAM-2/Mac-1 [16], syncytin-1/ASCT2 [17] and CD47/SIRPα [18,19] play important roles in cell recognition and differentiation. DCSTAMP (dendritic cell-specific transmembrane protein) has been uniquely identified as a master regulator of fusion in osteoclast and foreign body giant cells (FBGCs) [20–22]. Mensah et al. (2010) demonstrated that DC-STAMP surface expression levels varied within the precursor population and identified pre-OCs exhibiting low DC-STAMP expression as having the greatest fusion potential [23]. Cadherins, involved in homotypic cell–cell contacts, have been implicated as mediators of fusion for multiple cell types [24–26]. An increasing number of cellular immunology studies have explored Ecadherin function and regulation in activated macrophages and dendritic cells [14,25,27]. Interleukin-4 (IL-4) stimulated macrophages, which fuse to become multinucleated foreign body giant cells (FBGCs), upregulate E-cadherin and DC-STAMP expression early in the differentiation process [28]. In addition, multiple studies have implicated M- and N-cadherin during myoblast fusion and differentiation [29–32]. We wondered if similar mechanisms played a role in pre-osteoclast fusion as very few studies to date have examined cadherins during osteoclastogenesis. E-cadherin presence was established in marrow cell cultures and blocking E-cadherin function significantly reduced TRAP+ multinucleated cell numbers. Mbalaviele et al. also suggested that E-cadherin could participate in mature osteoclast resorptive functions [33]. While this study made a clear link between E-cadherin and osteoclast maturation, the timing of E-cadherin expression and the exact mechanism of action during osteoclast differentiation remain to be determined. We wanted to determine whether E-cadherin expression participated in precursor fusion competence and/or was a necessary mediator of the actual fusion event. This study sought to complement end-point population assays with live, single-cell analysis. Live-cell imaging was utilized to examine population dynamics while exploring individual cell morphology and behavior during phases of proliferation and fusion. We report here that E-cadherin is expressed during early stages of osteoclastogenesis. We show that endogenous surface expression of E-cadherin is visible at sites of cell–cell contact and membrane extensions in both RAW 264.7 cells and primary BMDMs. In addition, blocking E-cadherin function prior to peak fusion periods significantly reduced multinucleated cell formation in an NFATc1-dependent manner. This suggests that Ecadherin supports the development of fusion-competent precursor cells that utilize migratory protrusions to mediate pre-osteoclast fusion. 2. Materials and methods 2.1. Cell culture and osteoclast formation The RAW 264.7 murine macrophage cell line, obtained from the American Type Culture Collection (ATCC, VA, USA), was cultured in 5% CO2 and 37 °C conditions in alpha-Modified Eagles Medium (α-MEM) containing 10% heat-inactivated fetal bovine serum (Wisent Inc., QC, Canada). Confluent flasks were subcultured into 24-well or 6-well tissue culture plates at an average cell density of 3.0 × 104 and 1.5 × 105 cells/ well, respectively. Soluble GST-RANKL, produced with BL21 E. coli transformed with a pGEX-2T RANKL vector (a gift from Morris Manolson, University of Toronto), was added at a final concentration of 20– 30 ng/ml to each well. Fresh RANKL-containing medium was replaced every 48 h. For E-cadherin blocking assays, 3.0 × 104 cells/well were cultured in 24-well plates with 25 ng/ml RANKL along with 20 μg/ml of either rat IgG2b control isotype (Sigma Aldrich Ltd., ON, Canada) or ECCD-1, mentioned hereafter as E-cadherin bAb (anti-E-cadherin blocking antibody; Enzo Life Sciences Inc.). Treatments started at the time of cell plating

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and lasted for the whole duration of the experiment, unless otherwise stated.

2.2. BMDM isolation and differentiation Bone marrow cells were isolated from femurs and tibias of 6– 8 week-old wild-type BALB/c mice (obtained from Dr. Bebhinn Treanor, University of Toronto) as previously described [34]. The bone marrow cell pellet was treated with red blood cell (RBC) lysis buffer (Sigma Aldrich Ltd.) before being plated overnight in α-MEM containing 10% FBS, 1% penicillin/streptomycin (Wisent Inc.) and 25 ng/ml M-CSF (Peprotech, NJ, USA). Following the 24-h incubation, non-adherent cells were collected, counted and plated on coverslips at 8.0 × 104 cells/well in 24-well plates. Cells were stimulated with M-CSF (50 ng/ml) for 3 days prior to combined M-CSF (50 ng/ml) and RANKL (100 ng/ml). The medium was changed every three days and we were able to observe osteoclasts within 3–4 days after initial RANKL addition.

2.3. RT-qPCR analysis Total RNA extraction was performed using RNeasy mini kit (Qiagen, ON, Canada). The purity and quantity of RNA was determined with a NanoDrop ND-100 Spectrophotometer (Thermo Fisher Scientific Inc.) prior to cDNA synthesis. 1 μg of total RNA was used to generate cDNA using the SuperScript III First-Strand Synthesis SuperMix for quantitative RT-PCR (Invitrogen, ON, Canada). The primer sets used are shown in Table 1: The geometric mean from housekeeping genes, Gapd, Pgk, Rpl13a, Hprt1 (RealTime Primers, PA, USA) was used to normalize the data. The 25 μl qPCR reaction contained 10 ng of cDNA and utilized SYBRgreen (Bio-Rad, Ontario, Canada) as the detection method. The PCR reaction was performed with a DNA Engine Opticon System (BioRad). Primer sets were validated to have amplification efficiencies over 88%. Melting curve analysis was completed with each PCR run to test for nonspecific primer binding. Opticon Monitor software was used to determine the cycle threshold. Relative mRNA expression levels were calculated using the comparative Ct method (2−ΔΔCt) with GenEx qPCR analysis software (Bio-Rad). Three or four independent biological replicates were performed for each experiment and each reaction was run in triplicate.

2.4. Immunoblotting Total cell lysates were collected in radioimmunoprecipitation assay (RIPA) buffer (5 × buffer; Bio Basic Canada Inc.) containing freshly added protease and phosphatase inhibitors (Sigma Aldrich Ltd.). Protein quantification was completed with the DC Protein Assay (Bio-Rad) before 20 μg of each sample was loaded on 8% SDS-PAGE gels, transferred onto nitrocellulose membranes, and blocked with 5% milk for 1 h. Blots were incubated with primary antibodies overnight at 4 °C and with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, PA, USA) for 1 h. Primary antibodies used were: rat monoclonal antiDECMA-1 and mouse monoclonal anti-β-actin (Sigma Aldrich Ltd.) and rabbit polyclonal anti-E-cadherin (Proteintech, IL, USA). Densitometry analysis of protein bands from three biological replicates was performed using ImageJ (v1.49 software; NIH, USA). Table 1 Osteoclast-specific qPCR primer sequences (5′–3′). E-cadherin TRAP Cathepsin K DC-STAMP NFATc1

F-TCAGTTCCGAGGTCTACAC F-ACGGCTACTTGCGGTTTCA F-GAAGAAGACTCACCAGAAGCAG F-TACGTGGAGAGAAGCAAGGAA F-CCCGTCACATTCTGGTCCAT

R-CTTCAAATCTCACTCTGCCC R-TCCTTGGGAGGCTGGTCTT R-TCCAGGTTATGGGCAGAGATT R-ACACTGAGACGTGGTTTAGGAAT R-CAAGTAACCGTGTAGCTGCACAA

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2.5. Immunofluorescence and analysis Cells differentiated on coverslips until indicated days were washed with cold TBS + 1 mM Ca2+ before being incubated with a 1:50 dilution of anti-E-cadherin clone 36 (BD Biosciences, Canada) antibody for 10 min at 4 °C. Cells were fixed in 4% paraformaldehyde (PFA), blocked with 5% FBS and then incubated with Cy3-conjugated anti-mouse secondary (Jackson ImmunoResearch) for 1 h. Cells were permeabilized with 0.1–0.25% Triton X-100 with glycine, then counterstained for the following markers for 1 h in antibody binding buffer (TBS + 1 mM Ca2 +, 1% FBS): F-actin with Alexa Fluor 488-phalloidin (Invitrogen) and microtubules with mouse monoclonal alpha-tubulin (Sigma Aldrich Ltd.). Nuclei were stained with DAPI (4′,6-Diamidino-2Phenylindole, Dihydrochloride) for 10 min prior to mounting. Qualitative assessment of surface E-cadherin was performed with unedited images taken with identical parameters. Cells were considered positive for surface E-cadherin if approximately 50% of the plasma membrane had visible E-cadherin. NFATc1 staining required specific alterations to the basic staining protocol. Cells were permeabilized with 0.25% Triton X-100 containing 2% non-fat milk, 1% FBS and 0.2% BSA for 25 min, adapted from Yagi et al. [22]. A 1:200 dilution of NFATc1 clone 7A6 (Santa Cruz Biotechnology, Texas, USA) antibody was prepared in 2% non-fat milk, 1% FBS and 0.2% BSA antibody binding buffer and coverslips were incubated for 1 h. After washing, coverslips were incubated with Cy2-conjugated antimouse secondary prepared in the same antibody binding buffer for 1 h and mounted as above. For quantitative NFATc1 localization, CTCF (corrected total cell fluorescence) was determined using ImageJ v1.49 software (NIH, USA). CTCF was calculated with the following formula: Integrated Intensity − (area of interest ∗ mean background fluorescence). Samples were imaged using identical parameters and fluorescence intensity was compared between unaltered images. Total cell fluorescence and nuclear fluorescence were measured and used to calculate a ratio of nuclear: total fluorescence. For NFATc1 qualitative assessment, nuclei were scored as NFATc1-positive if signal intensity was markedly stronger in the nucleus compared to the cytosol. Cells that had an even distribution of nuclear:cytosolic signal were not included in the comparison. Fusion index (%) was determined by dividing the total number of nuclei present within osteoclasts (cells with ≥ 3 nuclei) by the total number of nuclei in the sample population. Ten randomly selected fields represented the sample population. Experiments were repeated a minimum of 3 times. 2.6. Production of a stable E-cadherin-GFP+ RAW 264.7 population Addgene plasmid 28009 insert was fully sequenced prior to use. Ecadherin-GFP chimera, a gift from Jennifer Stow (Institute for Molecular Bioscience, University of Queensland), was transfected using FugeneHD (Promega, WI, USA) according to manufacturer's instructions. Positive colonies were selected with 1 mg/ml of G418 for 3 weeks and then sorted twice using a BD FACSAria III to generate high-expressing populations. Transient transfection was utilized to characterize E-cadherin-GFP localization in RAW 264.7 cells. In brief, cells were transfected using Fugene-HD according to manufacturer's instructions 48 h after plating. Cells were differentiated with RANKL for a minimum of 30 h posttransfection before imaging. 2.7. Long-term live-cell DIC (differential interference contrast) and fluorescence microscopy RAW 264.7 cells were imaged using an inverted AxioObserver Z1 microscope (Carl Zeiss, NY, USA) and 25 mm glass bottom single well dishes (MatTek, MA, USA). A 20 × air objective or 40 × oil immersion lens were used for imaging and 6.0 × 104 cells/well were maintained

in α-MEM containing 20–30 ng/ml GST-RANKL in 5% CO2 and 37 °C conditions. Time-lapse movies were acquired over 10–40 h periods beginning from day 2, 3 or 4 of differentiation. Confocal images were taken using a WaveFX-X1 spinning disk confocal (Quorum Technologies, ON, Canada). MTrackJ Plugin for ImageJ (v1.49 software; NIH, USA) was used to determine the average migration rate of individual cells. Cells were followed for 3 h and movement was recorded every 5 min from the centroid of the cell. At least 100 cells/biological replicate were scored to determine overall rates. % Migration of a population was determined through visual analysis. Cells were followed for 3 h intervals and were considered migratory if they moved a full cell-length in the allotted time and retracted their tail. 2.8. Statistical analysis RT-qPCR data are presented as mean + SD. All other graphs are presented as mean ± SEM, unless otherwise indicated. Comparisons between groups were conducted using a Student's t-test. Comparisons between 3 or more treatment conditions were analyzed using a oneway ANOVA and Tukey's multiple comparisons post-test. All data was analyzed using GraphPad Prism version 6.0. All data were produced from 3 to 4 biological replicates and p b 0.05 was considered statistically significant, unless otherwise stated. Linear regression analysis was used to determine if individual proliferation curves were significantly nonzero. Slopes were compared by ANCOVA to assess whether they were significantly different (p b 0.05). 3. Results 3.1. E-cadherin localizes to distinct regions on the pre-osteoclast (pre-OC) cell membrane To better understand the role of E-cadherin in osteoclasts, we began with immunofluorescence analysis of surface E-cadherin over the course of osteoclastogenesis (Fig. 1). Individual RAW 264.7 cells (Fig. 1A) and primary murine BMDMs (Fig. 1B) were examined and compared for Ecadherin surface localization. In both cell types, surface E-cadherin was observed at sites of cell–cell contacts (Fig. 1, denoted with arrowheads) and at polarized regions of cells, suggesting an involvement in dynamic membrane protrusions (Fig. 1, indicated with *). A substantial proportion of pre-OCs had surface E-cadherin along their entire periphery. Remarkably, larger multinucleated cells from differentiated RAW and BMDM populations consistently showed negligible surface E-cadherin whereas smaller pre-OCs (≤ 3 nuclei), with prominent surface Ecadherin, were visible throughout differentiation. This indicates that a subset of pre-OCs maintain surface E-cadherin throughout osteoclast differentiation. From these observations we became interested in examining the relationship between E-cadherin surface expression, particularly at cell–cell contact sites, and osteoclastogenesis. 3.2. E-cadherin expression is highest during early stages of osteoclastogenesis To complement our initial observations, we examined the expression profile of endogenous E-cadherin under normal conditions for RANKL-stimulated osteoclastogenesis. First, we quantified the proportion of RAW 264.7 cells expressing surface E-cadherin throughout OC differentiation. The percentage of unstimulated control cells with surface E-cadherin for days 1–3 of culturing was calculated as 97 ± 0.6%, 91 ± 1.7% and 92 ± 0.8% respectively. Interestingly, the pre-OC population 1 day post-RANKL addition presented a similar proportion of cells with surface E-cadherin (96 ± 0.5%) as compared to unstimulated 1 day control cells (p = 0.99; Fig. 2A). Pre-OC populations differentiated with RANKL for 2 days displayed a significantly smaller number of Ecadherin positive cells (51 ± 3.4%) compared to unstimulated 2 day control levels (p b 0.001). At 3 days post-RANKL addition a striking

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Fig. 1. E-cadherin is present on the pre-osteoclast cell surface and concentrates at sites of cell–cell contact and at membrane protrusions. (A) RAW 264.7 cells were cultured with RANKL and (B) murine BMDMs were differentiated with M-CSF + RANKL for the indicated time periods. Cells were immunostained for external E-cadherin (red) and were counterstained with phalloidin (green) for F-actin and DAPI (blue) for nuclei. Arrowheads indicate regions of E-cadherin accumulation between contacting cells. (*) Identify E-cadherin at polarized regions of plasma membrane. Dashed lines indicate cell periphery of multinucleated osteoclasts. Images are a single 0.3 μm confocal slice and scale bars represent 20 μm.

reduction was observed with only 26 ± 4.4% of cells expressing surface E-cadherin compared to unstimulated 3 day control levels (p b 0.001). Overall, significantly fewer cells expressed surface E-cadherin as OC differentiation time increased. In addition, we examined whether the pattern of E-cadherin surface expression reflected changes in total E-cadherin transcript and protein levels. To provide context, TRAP and Cathepsin K mRNA levels increased significantly over 3 days in RANKL-stimulated RAWs (data not shown). TRAP and Cathepsin K participate in osteoclast resorption activities and are routinely used as markers of osteoclast maturation [3,35]. DCSTAMP, which is a known fusion mediator [20,21,36], is required during the early stages of differentiation. Despite this, DC-STAMP transcript levels followed an expression pattern that was similar to the mature markers (data not shown). Interestingly, E-cadherin expression did not increase with OC differentiation time (Fig. 2B). Instead, transcript levels were highest in control and 2d samples and exhibited significantly lower expression during day 3, when the majority of competent cells were fusing (see Fig. 1). E-cadherin protein was identified as a doublet band at 120 kDa and 100 kDa and was present throughout osteoclast differentiation, with greatest expression within 2 days of RANKL

stimulation (Fig. 2C). Unstimulated RAW 264.7 cells had a strong baseline expression of E-cadherin whereas a non-significant increase in E-cadherin protein was observed in 2d differentiating populations. E-cadherin protein began to decrease by day 3 and densitometry confirmed that 4d levels were significantly lower compared to the unstimulated control population. Both mRNA and protein expression data suggests a role for E-cadherin during early stages of osteoclast differentiation. The decline in E-cadherin transcript and protein expression was consistent with the decrease in the proportion of cells expressing surface Ecadherin during a 3-day differentiation period. This led us to question whether surface E-cadherin persistence and/or disappearance could be indicative of the differentiation state of individual pre-OC cells. To investigate this, we counterstained pre-OC cells for nuclear NFATc1 in RAWs and primary BMDMs (Fig. 2D–E). NFATc1 (nuclear factor of activated T-cells, cytoplasmic 1) is the master transcription factor of osteoclastogenesis, making it an acceptable marker of OC differentiation [36– 38]. Fluorescent nuclear NFATc1 signal could be distinguished as early as day 2 and maximally at days 3 and 4 of differentiation in both RAW 264.7 and primary BMDM cells (data not shown). RANKL-stimulated

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Fig. 2. E-cadherin expression is highest during the early stages of osteoclastogenesis. (A) The surface E-cadherin positive RAW 264.7 population (%) was quantified over a 3-day period. Approximately 300 cells/replicate were scored and a one-way ANOVA with Tukey's multiple comparisons test was performed. Treatments considered statistically different (p b 0.01) are represented with different letters (a–c). (B) RT-qPCR analysis of E-cadherin expression was performed over a 3-day time course with RAW 264.7 cells. Signal intensities were normalized to the geometric mean of a panel of housekeeping genes and fold change was determined relative to unstimulated control levels. Data are plotted as mean + SD (*p b 0.05; n = 3). (C) Western blot analysis over a 4-day time course with RAW cells was performed in triplicate. Signal intensity was normalized to β-actin and fold change was determined relative to control (*p b 0.05). (D) RAW 264.7 and (E) murine BMDMs were immunostained for external E-cadherin (red), total NFATc1 (green) and nuclei (blue) at the indicated days. Dashed lines indicate cell periphery and arrowheads highlight cells with strong surface E-cadherin. Cells were visually assessed for presence of both surface E-cadherin and nuclear NFATc1. Scale bars represent 20 μm.

pre-OCs from 2d and 3d populations were examined. Cells visibly expressing surface E-cadherin typically had lower to no discernable nuclear NFATc1 (see cells marked by arrowheads). This pattern was maintained when E-cadherin-positive mononuclear cells were examined at day 3, when large multinucleated osteoclasts had formed. This data suggests that a more differentiated state, as evidenced by greater NFATc1 nuclear accumulation, correlates with a decrease in surface E-cadherin presentation. 3.3. Blocking E-cadherin function significantly disrupts multinucleated OC formation and differentiation The above E-cadherin mRNA and protein expression data indicated that E-cadherin was actively regulated during osteoclastogenesis. To identify a functional role for E-cadherin, we began by asking whether a lack of E-cadherin could interfere with pre-osteoclast fusion. Our attempts at siRNA and shRNA knockdown of E-cadherin in RAW 264.7 cells proved unsuccessful (data not shown), leading us to address Ecadherin's role in OC fusion and differentiation with functional blocking antibodies. These antibodies have been successfully used to study Ecadherin function in IL-4-induced multinucleated giant cell formation [28]. We approached this by adding functional blocking antibody (Ecadherin bAb) to RAW 264.7 cells for time intervals that correlated

with early (1–2d) or late (3–4d) differentiation (Fig. 3A). The fusion index (%) was determined at day 4 for each treatment condition and cells with 3 or more nuclei were considered osteoclasts (Fig. 3B). We previously determined that the time of IgG isotype Ab addition did not affect OC fusion levels (data not shown), which allowed us to compare E-cadherin bAb treatments to a single control population. We confirmed that addition of 20 μg/ml of E-cadherin bAb for 4 days (E-cadherin bAb 1–4d) resulted in a significant reduction in fusion (approximately 50%) when added at the onset of differentiation, compared to isotype Ab control treatment (Fig. 3). To determine more precisely when E-cadherin was acting during osteoclastogenesis, we treated early and late-stage differentiating OCs with the same functional blocking Ab. The fusion index was also reduced by 50% in populations that had been treated with E-cadherin bAb during the first 2 days (E-cadherin bAb 1–2d) of OC differentiation (Fig. 3B). No significant decrease in overall fusion was observed if blocking antibody was added during the final 2 days (E-cadherin bAb 3–4d) of differentiation (Fig. 3B). Thus blocking E-cadherin function, specifically during the early stages (1–2d) of osteoclast differentiation, negatively impacts endpoint fusion levels of a differentiating OC population. Next, we evaluated whether the significant disruption of OC fusion levels in E-cadherin bAb-treated populations could be explained by a delay in OC-specific gene up-regulation. Transcript levels of TRAP,

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Fig. 3. Blocking E-cadherin function significantly impairs OC fusion. RAW 264.7 cells were differentiated in the presence of rat IgG isotype control or a functional blocking antibody against E-cadherin. (A) Populations were exposed to blocking antibody during early differentiation (1–2d, 1–4d) or late differentiation (3–4d) to determine effects on OC fusion levels. Representative examples of endpoint (4d) populations used to determine the fusion index are shown. Nuclei (pseudo-colored cyan) and alpha-tubulin (gray) are pictured. Dashed outlines delineate the boundaries of large multinucleated cells. Scale bars represent 20 μm. (B) Fusion Index (%) was calculated from 3 independent samples. Data are plotted as mean + SEM, with (*) p b 0.05 considered significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Cathepsin K and DC-STAMP were compared between E-cadherin bAbtreated populations and IgG isotype-treated populations after being normalized to the baseline expression of unstimulated RAWs (Fig. 4A). RT-qPCR analysis was performed with 2d cultures to investigate if early OC-specific gene expression was affected immediately following blocking antibody treatment. Each gene had dampened expression compared to gene levels observed in cells treated with control IgG (Fig. 4A), suggesting that blocking E-cadherin function delays the differentiation program of RANKL-stimulated RAW cells. Our attention turned to NFATc1, the master OC transcription factor, which regulates TRAP, Cathepsin K and DC-STAMP expression [36–38]. NFATc1 expression was also modestly, yet significantly, lower in E-cadherin bAb-treated

populations compared to IgG-treated control populations (Fig. 4B). Nuclear translocation of active NFATc1 is necessary for transcriptional initiation of not only OC-specific genes, but also for the auto-amplification of NFATc1 as well [37,38]. For these reasons, we examined whether addition of E-cadherin bAb correlated with changes in the localization of NFATc1 protein. Total cellular NFATc1 fluorescence intensity was initially measured from populations treated with either E-cadherin bAb or IgG Ab for 2 days. We confirmed that E-cadherin bAb- and IgG isotypetreated cells had no significant difference in total NFATc1 (Fig. 4C). However, when nuclear NFATc1 fluorescent signal intensity was compared to total NFATc1 signal intensity, the IgG isotype control populations had nearly 30% more nuclear NFATc1 compared to E-cadherin

Fig. 4. Blocking E-cadherin function delays early OC differentiation. (A, B) RT-qPCR analyses of the osteoclast-specific genes, TRAP, Cathepsin K, DC-STAMP and master transcription factor NFATc1, were performed at day 2 of differentiation for the indicated treatment conditions with RAW 264.7 cells. Signal intensities were normalized to the geometric mean of a panel of housekeeping genes and fold change was determined relative to unstimulated control levels. Data are plotted as mean + SD (*p b 0.05; n = 3). (C) Corrected total cell fluorescence of total NFATc1 levels at day 2 of differentiation for IgG isotype control and blocking antibody-treated samples were measured with ImageJ. (D) Representative immunofluorescent images of total NFATc1 used for fluorescence intensity measurements are shown. Scale bar represents 20 μm. (E) A ratio of nuclear NFATc1 fluorescence intensity to total NFATc1 cell fluorescence was calculated for IgG isotype control and blocking antibody-treated samples. Data are mean + SEM, with (*) p b 0.05 considered significant.

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bAb-treated populations (Fig. 4D, E). Taken together, impaired osteoclast formation and lower OC gene expression resulting from 2 days of incubation with E-cadherin bAb suggests that early E-cadherin activity strongly impacts osteoclast differentiation. 3.4. Blocking E-cadherin impairs migration, a necessary component for successful pre-OC fusion Next, population dynamics during prolonged exposure to E-cadherin blocking antibody were explored (Fig. 5). Previous research has established Rac1-mediated migration as a requisite part of osteoclast and foreign body giant cell (FBGC) differentiation [39–41]. In epithelial cells, disruption of E-cadherin expression and surface availability can be responsible for dramatic changes in migratory behavior [42]. We questioned whether a similar relationship between E-cadherin and migration was acting in RANKL-stimulated RAW 264.7 cells and could contribute to the significant reduction in OC fusion levels. For our study, four time intervals were chosen to examine the migratory behavior of

IgG-treated and E-cadherin bAb-treated RAW cells. The results from Figs. 3 and 4 demonstrated that after 2 days of E-cadherin bAb treatment both early OC gene expression and late stage OC formation were impacted. As a continuation of these observations, our analysis began at 48 h to determine if blocking E-cadherin activity could result in additional changes to long-term pre-OC population behavior. Fig. 5A shows DIC frames from movies that are representative of three independent experiments. In control conditions, mid-sized osteoclasts (4–9 nuclei) were visible by 60 h, whereas E-cadherin bAb-treated populations had minimal multinucleated cells. The average migration speed of individual cells was calculated to determine whether impaired migration, and therefore a diminished ability to locate a fusion-competent partner, could account for the difference in fusion levels. Individual mononuclear and binuclear pre-OC cells from 3d RANKL-stimulated populations were tracked for 3 h but no measurable difference in speed was found (Fig. S1). We concluded that blocking E-cadherin function did not affect the speed of migrating cells. However, this data was limited to the subpopulation of cells actively moving during the chosen time interval.

Fig. 5. Migration is a necessary component for successful pre-osteoclast fusion. (A–C) RANKL-stimulated RAW 264.7 cells were treated with IgG isotype control or E-cadherin functional blocking antibody for 48 h prior to imaging. Treatments continued for the duration of the experiment. (A) DIC frames provide a snapshot of population dynamics at time points chosen for further analysis. (*) Indicate osteoclasts with at least 3 nuclei. (B) % Migration of the total cell population per field was calculated at four time intervals spanning the entire period of differentiation. Data are presented as mean ± SEM (*p b 0.05) from multiple positions of 3 independent movies. (C) Linear regression analysis of relative proliferation spanning 48– 84 h of differentiation for each treatment condition is shown. Data are presented as mean ± SEM from multiple positions of 3 independent movies. Proliferation trends were compared with ANCOVA (*p b 0.01). (D) Fusion phenotype categories were identified through DIC observation. Examples of fusion at the leading edge (arrowhead) and fusion at an active protrusion (arrow) are shown. (E) Each fusion category (%) was quantified from 3 independent DIC movies and over 200 fusion events. Data are plotted as mean + SEM (*p b 0.01). (F–H) RAW cells were initially stimulated with RANKL for 48 h. After 48 h, media and RANKL were replaced and Wiskostatin or DMSO was added for a maximum of 24 h. (F) Arrowheads indicate fusion at the leading edge in representative DIC frames. (G) % Migration was determined with 100 cells/replicate utilizing multiple positions from the DIC movies. (H) Fusion index (%) was calculated from fixed-cell populations and a representative image of each condition is shown in (F), with actin (gray), nuclei (cyan) and dashed lines indicating cell periphery. Data represents mean + SEM of 3 independent experiments. A Student's t-test was performed (G, H) and * indicates p b 0.001. Scale bars (A, D, F) represent 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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When analysis was directed to population-level differences in migratory cells, a pattern emerged. From 48 to 51 h after treatment, the percent cell migration was comparable between IgG-treated (47 ± 3.2%) and E-cadherin bAb-treated (54 ± 2.0%) populations (p = 0.21; Fig. 5B). Similarly, from 60 to 63 h post-treatment, an average of 42 ± 1.6% IgG-treated and 47 ± 2.1% E-cadherin bAb-treated cells were migratory (p = 0.71). However, at 72 h onward, when the majority of osteoclast fusion occurs, the proportion of cells migrating in each condition was significantly different. At 72–75 h an average of 55 ± 2.4% IgG-treated cells were migratory compared to 45 ± 1.9% of Ecadherin bAb-treated cells (p = 0.025; Fig. 5B). At 84–87 h the trend continued with 60 ± 0.9% of IgG-treated control cells compared to 39 ± 1.3% E-cadherin bAb-treated cells displaying migratory behavior (p b 0.001). This data shows that blocking E-cadherin function impacts the migratory behavior of a population, particularly during peak periods of fusion. In addition, larger numbers of mononuclear cells were observed in E-cadherin bAb-treated populations from 72 to 84 h, compared to IgG isotype control populations (Fig. 5A). It was unclear whether this was the result of fewer fusion events or was the consequence of continued proliferation. The transition from a proliferative phase to one of terminal differentiation, involving multinucleation, has been well documented in primary osteoclast precursor cells [43,44]. Numerous studies in epithelial cells have demonstrated E-cadherin is involved in contact-dependent growth suppression [45,46]. For these reasons, it was necessary to determine if E-cadherin bAb addition could contribute to protracted cell growth in RAW 264.7 populations after RANKL activation. To investigate this, IgG- and E-cadherin bAbtreated populations were compared for relative proliferation (Fig. 5C). The number of nuclei per field was determined every 6 h from 48 to 84 h at multiple positions within the imaging well. Linear regression analysis was performed to interpret the proliferation trends of IgGtreated populations (R2 = 0.99, F(1,5) = 496.6, p b 0.001) and Ecadherin bAb-treated populations (R2 = 0.97, F(1,5) = 230.4, p b 0.001). When compared, regression lines were determined to be significantly different (F(1,10) = 11.6, p b 0.01). Notably, the higher proliferation maintained at later time points for E-cadherin bAb-treated populations correlated with the significant decline in percent cell migration determined in Fig. 5B. This suggests that the extended proliferation observed with E-cadherin bAb-treated samples was partly responsible for the reduced proportion of migrating cells. To summarize, prolonged proliferation coupled with decreased cell migration during the late stages (days 3–4) of differentiation appear to contribute to the reduced OC fusion levels observed in E-cadherin bAb-treated populations. The above migration data was compelling because fusion at the leading edge of migrating pre-OCs was frequently observed during our long-term imaging of OC differentiation (Fig. 5D; indicated with an arrowhead). Fusion could be separated into three major categories during normal RANKL-stimulated osteoclastogenesis. Irrespective of multinucleation status, 64 ± 1.6% of pre-OC cells fused at the leading lamella for one or both of the cells involved (p b 0.001; Fig. 5E; see Movie 1). A second fusion category involving active membrane extensions/protrusions could account for 23 ± 4.2% of all fusion events (Fig. 5D; denoted with an arrow). Rare events (14 ± 2.8%) were pooled into an “other” category and included fusion with a stationary cell and fusion with the tail of a migratory cell. These fusion categories show that the likelihood of fusion between pre-OCs increases when cells are migratory. Next, Wiskostatin, an inhibitor of actin nucleation and branching, was employed to examine the relationship between migration and fusion during OC differentiation. RAW 264.7 populations were differentiated with RANKL for 48 h prior to incubation with Wiskostatin or DMSO for an additional 20 h. Representative frames from DIC movies show DMSO-treated cells maintaining leading edges and participating in numerous fusion events (Fig. 5F, Movie 2). In Wiskostatin treated samples, cells were incapable of producing a leading edge (Fig. 5F, Movie 3).

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Attention was given to cells in close proximity to determine whether basal levels of fusion could occur in spite of Wiskostatin addition. Fusion events were rare, regardless of cell contact prior to Wiskostatin addition. The final panels of Fig. 5F are representative of the fixed populations used to determine the fusion index of each population. Both fusion ability (3.7 ± 0.3%; t(4) = 13.07, p b 0.001) and migration capacity (3.2 ± 0.1%; t(4) = 25.24, p b 0.001) were severely reduced in Wiskostatin-treated samples (Fig. 5G–H). This data serves to reinforce the relationship between pre-OC migration and fusion and supports the observations made with E-cadherin bAb-treated populations in Fig. 5A, B. 3.5. E-cadherin overexpression transiently enhances osteoclast differentiation RAW 264.7 cells stably expressing E-cadherin-GFP [47] were examined over a 4-day differentiation period to continue our analysis beyond blocking E-cadherin function. E-cadherin-GFP was observed on the cell surface, at sites of cell–cell contact (arrowheads) and at regions where active membrane ruffling (*) was visible (Fig. 6A). These findings corroborate the fixed-cell results of Fig. 1. Interestingly, we were unable to observe E-cadherin-GFP accumulating at the site of fusion between two differentiating pre-OCs (data not shown), suggesting E-cadherin does not mediate the actual fusion process. Since blocking E-cadherin function impaired OC differentiation (Fig. 3), it was necessary to determine if overexpression of E-cadherin could conversely enhance OC differentiation. We began by determining the fusion index of pre-OC populations stably expressing E-cadherin-GFP at days 2 and 3 of differentiation (Fig. 6B, C). A small but non-significant increase in fusion levels was observed between 3d populations of E-cadherin-GFP and GFP-only stably expressing RAWs. However, if the populations were assessed at an earlier time point (day 2) then overexpression of Ecadherin-GFP increased the fusion index. On average, the fusion index of 2d E-cadherin-GFP overexpressing populations was 5.5-fold more than the GFP-expressing controls. It was not uncommon to observe osteoclasts with 4 or more nuclei already present in 2d E-cadherin-GFP stable populations, while it was rare in GFP-alone control cells (Fig. 6B). In addition, a time course of E-cadherin-GFP expression levels was examined (Fig. 6D). Interestingly, E-cadherin-GFP expression was similar to the pattern of endogenous E-cadherin observed in RAW 264.7 cells (Fig. 2C). That is, E-cadherin-GFP levels were highest in control populations but were significantly reduced with increased exposure to RANKL. However, unlike endogenous E-cadherin expression, degradation of E-cadherin-GFP was already apparent by day 2. This earlier loss in total E-cadherin-GFP was consistent with the accelerated onset of OC fusion depicted in Fig. 6B–C and mirrored the reduction of endogenous E-cadherin that was observed when pre-OCs began fusing at day 3 of differentiation (Fig. 2C). Furthermore, analysis of 2d nuclear NFATc1 fluorescent signal intensity demonstrated greater intensity levels for E-cadherin-GFP expressing populations (Fig. 6E). The Ecadherin-GFP expressing population had a 20% increase in nuclear NFATc1 when compared to GFP-only controls. This supports the earlier appearance of multinucleated OCs in 2d E-cadherin-GFP populations and implies that the rate of OC differentiation was transiently accelerated. To summarize, the increased rate of osteoclastogenesis observed with E-cadherin-GFP expressing populations suggests that E-cadherin, likely through early cell–cell contacts, influences the progression of osteoclast differentiation. 4. Discussion In the present study, we have demonstrated a role for E-cadherinbased signaling at the onset of OC differentiation. Our results indicate that E-cadherin activity influences multiple aspects of osteoclastogenesis including OC-specific gene expression, reduction of early pre-OC expansion and transition to a period of migration, fusion and terminal differentiation (see model; Fig. 7).

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Fig. 6. E-cadherin overexpression contributes to an earlier onset of OC fusion. (A) RAW 264.7 cells were transfected with E-cadherin-GFP after 48 h of RANKL stimulation and were imaged 30 h after transfection. Cells were imaged for 12 h with DIC and fluorescence microscopy. Arrowheads indicate areas of E-cadherin accumulation at cell–cell contact sites. (*) Identify Ecadherin-GFP presence on polarized regions of plasma membrane. (B) GFP- or E-cadherin-GFP-expressing stables were immunostained for nuclei (pseudo-colored cyan) and alphatubulin (gray) after 2 days of RANKL treatment. Scale bar represents 20 μm. (C) Fusion Index (%) of 2d and 3d differentiated GFP- or E-cadherin-GFP-expressing populations was compared. A Student's t-test was performed separately for each day of differentiation (*p b 0.05). (D) Immunoblot analysis of E-cadherin-GFP over a 4-day differentiation time course was performed in triplicate. Signal intensity was normalized to β-actin and fold change was determined relative to control (*p b 0.05). (E) Nuclear NFATc1 fluorescence intensity was calculated relative to total NFATc1 cell fluorescence at day 2 for GFP- and E-cadherin-GFP stable populations. Data are mean + SEM, with (*) p b 0.05 considered significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

We have identified E-cadherin transcript and protein expression decreasing in a time-dependent manner after 48 h of RANKL-stimulated OC differentiation using RAW 264.7 cells. In addition, we determined

that the presence of surface E-cadherin persisted on small (1–3 nuclei) OC precursor cells and was substantially reduced in multinucleated, NFATc1-positive osteoclasts. Similar trends were observed when

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Fig. 7. Schematic of osteoclast differentiation mediated through E-cadherin. (Days 0–1) Pre-osteoclasts with high levels of E-cadherin and low nuclear NFATc1 are stimulated with RANKL. These undifferentiated cells are dispersed with few intercellular contacts; proliferation is induced. (Day 2) Osteoclast precursor numbers have increased and cell–cell contact via Ecadherin is more frequent. Proliferation reduces as the proportion of migratory pre-OCs increases. NFATc1-positive nuclei are visible, while E-cadherin surface expression begins to decrease. (Days 3–4) Osteoclast formation is characterized by multiple fusion events and minimal proliferation. Mono- and multinucleated fusion-competent cells have NFATc1positive nuclei and migrate toward potential fusion partners.

Mbalaviele et al. [33] utilized primary marrow cultures and stimulated osteoclastogenesis indirectly through addition of 1,25 dihydroxyvitamin D3 [33]. In this model, highest E-cadherin levels were observed at day 4, when TRAP+ mononuclear cells began fusing and levels decreased by day 6, when large OCs had formed. In contrast, in primary FBGC formation, E-cadherin up-regulation peaked within 6 h of IL-4 addition and high levels were maintained after 48 h [27]. Although different pathways and gene targets are activated during OC and FBGC differentiation [28,48], our combined work signifies an important role for E-cadherin is conserved in both OC and FBGC formation. Moreover, a study of IL-4-stimulated monocyte chemoattractant protein-1 (MCP-1) null macrophages suggested that atypical expression of matrix metalloproteinase-9 (MMP-9) and the E-cadherin/β-catenin complex could significantly reduce FBGC formation and differentiation [49]. According to this study, persistence of surface E-cadherin after 48 h of IL-4 stimulation identified an abnormal phenotype [49]. It was speculated that MMP-9 may be involved in external E-cadherin shedding, triggering internalization. This study, in combination with our data, significantly questions the likelihood that E-cadherin-based cell– cell attachment is involved in the fusion event. Indeed, the potential connection between E-cadherin and MMP-9 is intriguing because MMP-9 expression and activity has been shown to increase within 48 h of RANKL stimulation [50]. Since this correlates with the timedependent decrease in surface E-cadherin expression we observed in pre-OCs, it is possible that MMP-9 may play a similar role during OC differentiation. Examination of this relationship could be an interesting topic for future study. IL-4-mediated primary FBGC formation is significantly reduced by addition of E-cadherin neutralizing antibody [28]. The interval of Ecadherin bAb incubation time was varied to determine when Ecadherin influenced FBGC formation. As we observed with OC differentiation, addition of E-cadherin bAb immediately following IL-4 stimulation produced the greatest reduction in FBGC fusion levels, compared to control populations. Combined with our data, the evidence suggests Ecadherin functions early in the differentiation process, before the majority of cell–cell fusion, to impact both OC and FBGC differentiation. Conversely, E-cadherin bAb addition to mouse marrow cultures indicated that E-cadherin was required during late stages (days 4–6) of

osteoclastogenesis [33]. The additional variable of osteoblasts/stromal cells and their response to E-cadherin neutralizing antibody may account for such disparate results. Addition of E-cadherin bAb dampens osteoblast differentiation [51], which could potentially impact the progression of osteoclastogenesis. We used a homogeneous culture of RAW cells to study the effects of E-cadherin bAb treatment on pre-OC cell–cell contacts. Given that the in vitro model of osteoclastogenesis can influence the outcome of osteoclast differentiation, direct comparisons could not be made between these studies. However, we can conclude that both demonstrate a strong relationship between E-cadherin and OC differentiation. Unlike previous research [33], we chose to examine early OC-specific gene expression in response to blocking E-cadherin function. Analysis of E-cadherin bAb-treated populations identified a novel relationship; Ecadherin-based cell–cell contact had the ability to affect the OC differentiation program. Dampened expression of NFATc1, TRAP, Cathepsin K and DC-STAMP was observed after 2 days of RANKL treatment. We have also, for the first time, studied the effects of E-cadherin overexpression on OC differentiation. E-cadherin-GFP stably expressing populations showed earlier NFATc1 nuclear accumulation and OC formation, both of which indicate enhanced differentiation. Reduction of E-cadherin-GFP levels at the onset of fusion indicated that E-cadherin regulation was a RANKL-dependent process and supported the time-dependent decrease we observed with endogenous E-cadherin expression. Overall, examining OC differentiation beyond the usual parameters of the population fusion index and/or the number of TRAP+ cells allowed us to gain valuable insight into the role E-cadherin has during early OC differentiation. Since intercellular attachment and communication are important throughout osteoclast differentiation [52], it is conceivable that more than one type of cell–cell adhesion molecule could participate during the early period (days 1–2) of differentiation in which we have positioned E-cadherin function. Similar to our observations, blocking Mac1/Cd11b function with neutralizing antibody in BMMs resulted in a significant reduction of NFATc1 mRNA levels after 24 h of M-CSF and RANKL treatment [53]. Furthermore, ICAM-1/2 and Mac-1, proposed binding partners, localized to regions of cell–cell contact and multinucleated osteoclasts lost Mac-1 surface expression [16]. Redundancy in cell–cell recognition molecules may explain why blocking E-cadherin

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function resulted in a modest delay in OC-specific gene up-regulation and a 50% reduction in the fusion index. A recent study arrived at a similar conclusion after the in vivo activation and fusion of E-cadherindeficient macrophages was comparable to wildtype levels after Schistosoma mansoni challenge [54]. In summary, multiple coordinated players, including E-cadherin, are likely responsible for contact dependent responses during OC differentiation. Besides impairing OC differentiation and formation, we found that blocking E-cadherin-based cell–cell contact prolonged the proliferative phase of osteoclastogenesis. It has been established that terminal differentiation typically involves a period of precursor expansion, followed by cell cycle exit and then differentiation [11,55]. However, a recent study proposed that cell density and not prior proliferation, ultimately determined OC formation and differentiation [56]. Inhibition of DNA synthesis with hydroxyurea (HU) treatment for 48 h almost completely prevented ex vivo BMDM osteoclastogenesis but could be reversed if HU-treated pre-OCs were re-plated at increasing cell densities [56]. This is interesting as it implies that cell–cell contact directly enhances osteoclastogenesis. The same study showed that RANKL stimulation did not immediately cause cell cycle exit. Rather, cell division continued for the first 48 h of combined M-CSF and RANKL exposure [57]. Similarly, our data demonstrated E-cadherin expression remained high for the first 48 h of differentiation, presumably to aid in cell density-dependent osteoclast differentiation. It is tempting to hypothesize that blocking Ecadherin-based contacts (and the signaling events that follow) creates a situation where the pre-OC population misinterprets cell density cues, resulting in the continued proliferation of pre-OCs. Research concerning the relationship between N-cadherin-based cell–cell contact and myogenic differentiation has provided a possible link between cadherins, cell cycle withdrawal and terminal differentiation. Myoblasts, when plated at low density conditions to eliminate intercellular contacts, underwent normal differentiation when cultured on N-cadherin-Fc coated surfaces [30]. N-cadherin activation triggered the nuclear accumulation of the cyclin-dependent kinase (Cdk) inhibitors p21 and p27 and this was correlated with reduced cell proliferation (observed as a lack BrdU incorporation) [30]. It was proposed that Ncadherin functioned to regulate the proliferation-to-differentiation switch of precursor cells during myogenesis. Cyclin-dependent kinase inhibitors also contribute to cell cycle withdrawal during osteoclast differentiation [8,44]. In BMMs endogenous levels of p21 and p27 increased for the first 24 h of RANKL stimulation, with levels dropping after 48 h [44]. Remarkably, this trend correlates with the changes we observed in E-cadherin expression. Moreover, in vitro BMM differentiation from p21/p27 double knockout mice showed significant reduction in multinucleated osteoclast formation compared to wildtype, along with a failure to induce TRAP and Cathepsin K expression [8]. For these reasons Cdk inhibitors could provide a meaningful connection between E-cadherin ligation and cell cycle arrest. The expression of p27 is responsive to RANKL treatment in RAW 264.7 cells [57], making p27 an appealing candidate for further analysis. Lastly, we noted that extended proliferation resulting from blocking E-cadherin correlated with a decrease in the proportion of pre-OCs migrating and fusing. These observations support the growing literature that identifies monocyte and pre-OC migration as an essential element in osteoclast differentiation. The activities of Rho GTPases, regulators of the actin cytoskeleton, have been studied throughout OC differentiation [58]. For instance, the fusion efficiency of Rac1-null and Rac1/2double knockout osteoclast precursors was significantly impaired in both in vitro and in vivo osteoclastogenesis [39]. Similarly, severely compromised chemotactic migration toward both M-CSF and RANKL was also observed when Flna-null monocytes were compared against wildtype cells [40]. Osteoclast numbers could be restored if Flna (Factin crosslinking protein)-deficient pre-OCs were plated at a higher density to overcome migration limitations. Both studies convincingly demonstrate the importance of migration prior to pre-OC fusion and corroborate our observation that reduced migration is a key contributor

to the decreased fusion index of the E-cadherin bAb-treated populations. Several recent studies have employed long-term live imaging techniques to identify phenotypes suggestive of fusion-competent cells. In our study, we showed that Wiskostatin treated pre-OCs were incapable of forming a leading edge and displayed minimal fusion. Similarly, researchers studying actin dynamics during OC differentiation observed pre-OCs primarily fusing through cellular extensions (termed ‘fusopods’) located at the leading edge of one pre-OC and the tail of the other fusion partner. A second study using human monocytes found that mononuclear cells were primarily migratory, while highly multinucleated cells were more often immobile but produced many cellular extensions similar to our active protrusion category [59]. Also, they defined ‘broad contact surface’ as the most prevalent type of contact that preceded fusion, which appeared very similar to our leading edge type of fusion. These studies, including our own, are examples of how long-term imaging can reveal important behavioral and morphological details at the single-cell level within a sample population. In summary, this study has identified a role for E-cadherin during osteoclastogenesis that has not been previously described. E-cadherin did not appear to play an essential role during the OC fusion process. Instead, our data indicates that intercellular contact mediated through E-cadherin influences OC-specific gene induction and the transition to migratory and fusion-related behaviors. Each stage of osteoclastogenesis was captured using long-term imaging and provided us with a comprehensive look at pre-OC behavior. Live imaging reaffirmed the existing knowledge that cell–cell contacts and precursor migration are indispensible for osteoclastogenesis. Overall we have shown that manipulation of early cell–cell adhesion molecules during osteoclastogenesis has a significant impact on the osteoclast formation and differentiation potential of a population. Periods of differentiation involving early cell–cell recognition and contacts should continue to be explored as these surface molecules and downstream signaling elements could be therapeutic targets for managing bone remodeling diseases. Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. Author contributions CF and REH conceived and designed the study. CF performed the bulk of the experiments and analysis and wrote the first drafts of the manuscript. REH reviewed the results and edited the manuscript. Both authors approved the final version of the manuscript. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bone.2016.03.004. Acknowledgments We would like to thank Dr. He Song Sun. for critical reading of the manuscript and help with the production of stable E-cadherin-GFP RAW 264.7 cells. This project is funded by a Natural Science and Engineering Research Council grant (RGPIN 298538-09) to R.E.H. R.E.H. is a recipient of an Ontario Early Researcher Award. References [1] B.F. Boyce, Z. Yao, L. Xing, Osteoclasts have multiple roles in bone in addition to bone resorption, Crit. Rev. Eukaryot. Gene Expr. 19 (2009) 171–180. [2] R. Iwasaki, K. Ninomiya, K. Miyamoto, T. Suzuki, Y. Sato, H. Kawana, et al., Cell fusion in osteoclasts plays a critical role in controlling bone mass and osteoblastic activity, Biochem. Biophys. Res. Commun. 377 (2008) 899–904, http://dx.doi.org/10.1016/j. bbrc.2008.10.076. [3] W.J. Boyle, W.S. Simonet, D.L. Lacey, Osteoclast differentiation and activation, Nature 423 (2003) 337–342, http://dx.doi.org/10.1038/nature01658.

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