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. 2018 Apr 16;233(9):7497–7513. doi: 10.1002/jcp.26603

Transglutaminase activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics

Huifang Sun 1, Mari T Kaartinen 1,2,✉
PMCID: PMC13482107  PMID: 29663380

Abstract

Osteoclasts, bone resorbing cells, derive from monocyte/macrophage cell lineage. Increased osteoclast activity is responsible for bone destruction in diseases such as osteoporosis, periodontitis and rheumatoid arthritis. Transglutaminases (TGs), protein crosslinking enzymes, were recently found involved in osteoclastogenesis in vivo, however their mechanisms of action have remained unknown. In this study, we have investigated the role of TG activity in osteoclastogenesis in vitro using four TG inhibitors, NC9, Z006, T101, and monodansyl cadaverine. Our results showed that all TG inhibitors were capable of blocking the entire osteoclastogenesis process. The most potent of the inhibitors, NC9 when added to cultures at different phases of osteoclastogenesis, inhibited differentiation, migration, and fusion of pre‐osteoclasts as well as resorption activity of mature osteoclasts. Further investigation into the mechanisms revealed that NC9 increased RhoA levels and blocked podosome belt formation suggesting that TG activity regulates actin dynamics in pre‐osteoclasts. The inhibitory effect of NC9 on osteoclastogenesis as well as podosome belt formation was completely reversed with a Rho‐family inhibitor Exoenzyme C3. Microtubule architecture, acetylation, and detyrosination of α‐tubulin were not affected. Finally, we demonstrated that macrophages and osteoclasts expressed mRNA of three TGs:TG1, TG2, and Factor XIII‐A which were all differentially regulated in these cells during differentiation. Immunofluoresence microscopic analysis showed that all three enzymes co‐localized to podosomes in osteoclasts. Taken together, our data suggests that TG activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics and that this may involve contribution from all three TG enzymes.

Keywords: migration, osteoclastogenesis, podosomes, RhoA, transglutaminases

In this study, we show that chemical inhibition of transglutaminases (TGs) with NC9, Z006, T101 and MDC, blocks the entire osteoclastogenesis process. The most potent of the inhibitors, NC9, when added to cultures at different phases, inhibited differentiation, migration and fusion of pre‐osteoclasts as well as resorption activity of mature osteoclasts. The inhibition resulted in increased RhoA levels and blocked podosome belt formation suggesting that TG activity regulates actin dynamics in osteoclasts. Three TG enzymes, TG2, Factor XIII‐A and TG1, were discovered expressed in macrophages and mature osteoclasts where they localized to podosomes.

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1. INTRODUCTION

Bone resorption is orchestrated by osteoclasts which are large multinucleated cells derived from monocyte/macrophage lineage. The differentiation from monocytes/macrophages to mature osteoclasts is an intricate process involving a differentiation stage which is followed by a cell fusion and multinucleation stage. These complex events require both extensive interactions between cytokines and receptors, and dynamic rearrangement of cytoskeleton and plasma membrane (Yavropoulou & Yovos, 2008). Osteoclast differentiation requires two essential cytokines, macrophage colony‐stimulating factor (M‐CSF) and receptor activator of nuclear factor‐κB ligand (RANKL) (Teitelbaum, 2000). M‐CSF mediates proliferation, differentiation and survival of osteoclast precursors and promotes expression of RANK in osteoclast precursors, priming the RANK‐positive cells to respond to RANKL (Ross & Teitelbaum, 2005) which in turn induces sequential expression of NF‐κB, c‐Fos and NFATc1 (Boyce & Xing, 2008). NFATc1 is the master regulator of osteoclast differentiation and regulates osteoclast‐specific genes such as tartrate resistant acid phosphatase (TRAP) (Takayanagi et al., 2002) and cathepsin K (CTSK) (Matsumoto et al., 2004) as well as matrix metalloproteinase‐9 (MMP‐9) (Sundaram et al., 2007). With the continuing stimulation of RANKL, mononuclear TRAP‐positive (TRAP+) pre‐osteoclasts migrate toward each other and fuse to form multinucleated osteoclasts. This fusion process is regulated by two transmembrane proteins‐dendritic cell‐specific transmembrane protein (DC‐STAMP) and osteoclast‐stimulatory transmembrane protein (OC‐STAMP) (Yagi et al., 2005; Yang et al., 2008; Witwicka et al., 2015; Zhang, Dou, Xu, & Dong, 2014), both transcriptionally regulated by NFATc1 (Kim, Lee, Ha Kim, Choi, & Kim, 2008; Miyamoto et al., 2012). Unlike most non‐transformed cells relying on F‐actin stress fibers or focal adhesion plaques, cells from monocytic lineage such as dendritic cells, macrophages and osteoclasts develop podosomes to adhere and migrate with (Linder & Aepfelbacher, 2003). Podosomes are highly dynamic dot‐like structures, containing an F‐actin core and a surrounding F‐actin cloud, along with a variety of molecules such as integrins, kinases and small GTPases (Rho and Rac) (Jurdic, Saltel, Chabadel, & Destaing, 2006). On non‐mineralized substrates, podosomes in osteoclasts can self‐assemble into clusters, rings and finally form a stable peripheral actin belt (Jurdic et al., 2006). The assembly/disassembly of podosome superstructures allows osteoclast migration during fusion process and during bone resorption (Hu et al., 2011; Georgess, Machuca‐Gayet, Blangy, & Jurdic, 2014).

Transglutaminases (TGs; EC 2.3.2.13) are a family of thiol‐ and Ca2+‐dependent acyl transferases that catalyze the formation of a covalent bond between γ‐carboxamide groups of peptide‐bound glutamines and free amine groups (e.g., protein‐ or peptide‐bound lysines) (Iismaa, Mearns, Lorand, & Graham, 2009; Muszbek, Bereczky, Bagoly, Komaromi, & Katona, 2011; Eckert et al., 2014). Nine TG genes are present in humans, TG1‐7 and Factor XIII‐A (FXIII‐A), which are catalytically active enzymes. One family member, erythrocyte membrane protein band 4.2, is inactive as a transglutaminase (Eckert et al., 2014). So far, TG2 and Factor XIIIA have been shown involved in cell adhesion, migration (Akimov & Belkin, 2001), differentiation (Adany et al., 2001), and in function of monocytes and macrophages (Bagoly, Katona, & Muszbek, 2012; Chrobok, Sestito, Wilhelmus, Drukarch, & van Dam, 2017; Hodrea et al., 2010). TGs have been long reported expressed and found in bone and osteoblasts (Al‐Jallad et al., 2006, 2011; Cui & Kaartinen, 2015; Cui, Wang, Myneni, Hitomi, & Kaartinen, 2014; Heath, Downes, Verderio, & Griffin, 2001; Kaartinen, El‐Maadawy, Rasanen, & McKee, 2002; Nakano, Al‐Jallad, Mousa, & Kaartinen, 2007; Nurminskaya & Kaartinen, 2006; Piercy‐Kotb et al., 2012; Wang, Telci, & Griffin, 2011). Three recent studies by us and others suggest their role also in osteoclastogenesis and bone resorption (Kim et al., 2017; Mousa et al., 2017; Raghu et al., 2015). The work of Raghu et al. showed that FXIII‐A deficiency in mice led to reduced osteoclastogenesis in vivo and in vitro and that transglutaminase inhibitor, cystamine inhibited osteoclastogenesis in vivo (Raghu et al., 2015). Furthermore, our recent work showed that deletion of both TG2 and FXIII‐A gene expression in a double knockout mouse model resulted in severe osteopenia caused by increased bone resorption (Mousa et al., 2017). The increased osteoclastogenesis was also seen in double FXIII‐A/TG2 deficient monocytes in vitro demonstrating that the two enzymes negatively regulate osteoclastogenesis. However, similarly, as in the work of Raghu et al. (2015) a chemical inhibitor of TGs blocked osteoclastogenesis in our study, which was attributed to expression of TG1 in wild type and FXIII‐A/TG2 deficient monocytes and osteoclasts (Mousa et al., 2017). Although we did not see a bone phenotype in either, individual TG2 or FXIII‐A knockouts (Mousa et al., 2017), Kim et al. (2017) showed recently that TG2 deficient mice exhibited increased osteoclast number and lower trabecular bone mass in vivo and that siRNA knockdown of TG2 gave rise to increased osteoclastogenesis in vitro (Kim et al., 2017). The data from these studies strongly suggest that TGs are an important part of osteoclastogenic program and suggest an interplay between the three TGs in the process. The aim of this study was to investigate effect of TG inhibitors on osteoclastogenesis and to shed light on the mechanisms how they may exert their effect. We report that there are indeed three TGs expressed in macrophages and osteoclasts and that TG activity regulates differentiation, as well as migration and fusion of osteoclasts via affecting actin dynamics in these cells.

2. MATERIALS AND METHODS

2.1. Reagents and antibodies

MEM Alpha (αMEM) (12561–056), penicillin‐streptomycin, l‐glutamine, sodium pyruvate were from Gibco (Burlington, ON, Canada). Fetal bovine serum was from Hyclone (Waltham, MA). Human M‐CSF and human sRANK Ligand were from PeproTech (Rocky Hill, NJ). NC9 was synthesized by Gene Tech Inc (Indianapolis, IN) (Al‐Jallad et al., 2011). Monodansyl cadaverine (MDC) was from Sigma–Aldrich (St Louis, MO). Z006 and T101 were from Zedira GmbH (Darmstadt, Germany). RhoA inhibitor (Exoenzyme C3) (CT04) was from Cytoskeleton (Denver, CO). All other reagents unless otherwise specified were purchased from Sigma–Aldrich (Oakville, ON, Canada) or Fisher Scientific (Hampton, NH).

Mouse monoclonal TG1 antibody (E‐6) was from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal TG2 (Ab‐1) antibody was from Fisher Scientific. Rabbit anti‐mouse FXIII‐A (675–688 peptide sequence) (polyclonal antibody) was designed and generated by GenScript corporation (Piscataway, NJ) (Al‐Jallad et al., 2011). Mouse monoclonal acetylated α‐tubulin antibody (6‐11B‐1), rabbit polyclonal detyrosinated α‐tubulin antibody were from Abcam (Cambridge, MA). Rabbit polyclonal actin antibody, mouse monoclonal α‐tubulin antibody (DM1A), mouse monoclonal biotin antibody (BN‐34) were from Sigma–Aldrich. Rabbit polyclonal fibronectin antibody was from EMD Millipore (Billerica, MA). Mouse IgG1 kappa isotype control, mouse IgG2a kappa isotype control, Rabbit IgG isotype control, secondary antibodies Alexa Fluor® 568 and 488 conjugates, Alexa Fluor® 488‐phalloidin and DAPI (4′, 6‐diamidino‐2‐phenylindole) were from Thermo Fisher Scientific (Rockford, IL). Secondary antibody HRP‐linked anti‐mouse IgG was from GE Healthcare (Mississauga, ON, Canada). HRP‐linked anti‐rabbit IgG was from Cell Signaling (Whitby, ON, Canada).

2.2. Osteoclast differentiation from mouse bone marrow cells

Studies were compliant with McGill University guidelines established by the Canadian Council on Animal Care. Mouse bone marrow cells were collected from 6 to 10 weeks old C57BL/6 mice (from Jackson Laboratories) as described previously with minor modifications (Tiedemann et al., 2013). Briefly, bone marrow cells were plated at a density of 15 × 106 cells per 75 cm2 tissue culture flask and cultured overnight in αMEM supplemented with 10% FBS, 1% penicillin‐streptomycin, 1% l‐glutamine solution, 1% sodium pyruvate and 25 ng/ml M‐CSF. After 24 hr, non‐adherent cells were collected and plated at 5 × 104 cells/cm2 and incubated with 50 ng/ml M‐CSF. After 48 hr, the non‐adherent cells were washed out and adherent cells were considered to be M‐CSF‐dependent bone marrow‐derived macrophages and used as osteoclast precursors. Osteoclast differentiation was then induced by 50 ng/ml M‐CSF and 50 ng/ml RANKL for 5 or 6 days with two medium changes on day 2 and 4. The bone marrow macrophages were also treated with only 50 ng/ml M‐CSF for 5 or 6 days to serve as negative control cultures. Only joint treatment with M‐CSF and RANKL results in osteoclastogenenesis. TG activity inhibitor NC9, MDC and Z006 were dissolved in dimethylsulfoxide (DMSO), T101 in PBS. RhoA inhibitor (Exoenzyme C3) was dissolved in sterile water.

2.3. Cell viability assay

Cell viability in the presence of inhibitors was determined using the methyl‐thiazol tetrazolium (MTT) assay. Bone marrow macrophages were plated in 96‐well plates and incubated with M‐CSF and RANKL in the presence or absence of NC9, MDC, Z006, and T101. After 5 days, 0.25 mg/ml MTT reagent was added and incubated for 3 hr. Purple precipitates were dissolved in DMSO. The absorption was measured at 560 nm using a microplate reader (TECAN infinite F200 PRO, Männedorf, Switzerland). The experiments were independently repeated three times, each time in triplicate.

2.4. TRAP staining and cell counting

Bone marrow macrophages were plated in 96‐well plates and incubated with M‐CSF and RANKL in the presence or absence of different concentrations of NC9, MDC, T101, Z006 and 5 ng/ml Exoenzyme C3. Controls were treated with the drug vehicles. At the end points, cells were fixed with 3.7% formaldehyde and stained for TRAP with the TRAP Staining Kit (Cosmo Bio, Carlsbad, CA). Pre‐osteoclasts were identified as mononuclear TRAP+ cells and osteoclasts were identified as multinucleated (≥3 nuclei) TRAP+ cells. Cells were then stained with DAPI to facilitate counting nuclei. The experiments were independently repeated three times, each time in triplicate. For each experimental condition, five non‐overlapping images per well were taken under 10× objective to evaluate osteoclast differentiation.

2.5. TRAP activity assay

TRAP activity in the culture supernatant was measured with the TRAP Staining Kit (Cosmo Bio) as described previously with minor modifications (Li et al., 2016). Briefly, chromogenic substrate was dissolved with tartrate‐containing buffer and this reaction buffer was added to the culture supernatants in a 96‐well plate. The plate was then incubated for 3 hr in the dark at 37 °C and then read in a microplate reader at 540 nm (Molecular Devices SpectraMax M2e, Sunnyvale, CA). The experiments were independently repeated three times, each time in triplicate.

2.6. Osteoclastic resorption assay

Osteoclastic resorption assays were conducted on Corning® Osteo Assay Surface 24‐well plates coated with inorganic crystalline calcium phosphate (Tewksbury, MA). Bone marrow macrophages were plated and osteoclastogenesis was induced as described above. After 5 days, mature osteoclasts were cultured for further 5 days with M‐CSF and RANKL in the presence or absence of NC9. Resorption pits were visualized by light microscopy after modified von Kossa staining (Kartner et al., 2010) and quantified with ImageJ (NIH). The experiments were independently repeated three times, each time in triplicate. For each experimental condition, five images per well were taken under 5× objective to evaluate osteoclast resorption.

2.7. Migration assay

Cell migration was measured using a cell exclusion zone assay (Hulkower & Herber, 2011). Bone marrow macrophages were plated into Oris™ 96‐well plate with Oris™ cell seeding stoppers to prevent cells from attaching to the central zone with a diameter of 2 mm (Platypus Technologies, Madison, WI). Cells were cultured with M‐CSF and RANKL for 2 days and the stoppers were removed to reveal the exclusion zone into which pre‐osteoclasts were then allowed to migrate with treatment of M‐CSF and RANKL in the presence or absence of 20 μM NC9 for 12 hr. The exclusion zones were monitored under 5× objective and the number of migrated cells were quantified by counting cells that crossed into the void zone during the 12 hr. The experiments were independently repeated three times, each time in triplicate.

2.8. Assessment of podosome belt formation

Bone marrow macrophages were differentiated for 5 days with M‐CSF and RANKL in the presence or absence of 10 μM NC9 and 5 ng/ml Exoenzyme C3. When the osteoclast formed, cells were fixed with 3.7% formaldehyde and labeled with Alexa Fluor® 488‐phalloidin to visualize specific F‐actin structure‐containing podosome belts. The extent of podosome belt formation was quantified by counting the podosome belts versus the total number of osteoclasts and expressed as percentage. The experiments were independently repeated three times, each time in triplicate. For each experimental condition, three images per well were taken under 10× objective to evaluate osteoclast podosome belt formation.

2.9. Protein extraction and Western blotting

Cell lysates were prepared with ice‐cold cell lysis buffer containing 50 mM Tris (pH 7.5), 10 mM MgCl2, 0.5 M NaCl, and 2% Igepal, 1% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail and incubated for 30 min on ice. Cells were scraped and the lysates were further disrupted by passage through a 25G syringe needle for 30 times on ice and centrifuged at 14,000g for 15 min at 4 °C. Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific). For western blotting, 20 µg proteins were separated on 10% SDS–PAGE gels and transferred to PVDF membrane (Bio‐Rad, Mississauga, ON, Canada) followed by blocking with 5% nonfat milk in Tris‐buffered saline with 0.1% Tween 20 (TBST), and individual proteins were detected with specific antibodies as well as corresponding secondary antibodies conjugated with horseradish peroxidase. Bands were visualized using the ECL Plus kit (GE Healthcare), and chemiluminescence was detected using ChemiDoc™ Touch Imaging System (Bio‐Rad).

2.10. In vitro TG activity assay

In vitro TG activity assay was performed as described previously (Cui & Kaartinen, 2015). Briefly, cell lysate was incubated for 2 hr at 37 °C with 1 µg bovine plasma fibronectin (pFN), 2 mM 5‐(biotinamido) pentylamine (bPA), in a reaction buffer containing 1 mM dithiothreitol (DTT), 3 mM CaCl2, 10 mM Tris‐HCl (pH 8.0). After incubation, samples were analyzed by SDS‐PAGE and western blotting as above to visualize covalent bPA incorporation to pFN which reflects the TG activity present in the samples.

2.11. RhoA activity assay

GTP‐bound active RhoA was quantified using the RhoA Pull‐down Activation Assay Biochem Kit (Cytoskeleton) according to the manufacturer's protocol. Briefly, bone marrow macrophages were cultured with M‐CSF and RANKL in the presence or absence of 20 μM NC9 for 2 days. After the cell lysate collection and protein concentration measurement, GTP‐bound RhoA was pulled down from whole‐cell extracts using Rhotekin RBD beads. Affinity precipitates and whole‐cell lysates were analyzed by SDS–PAGE and western blotting as above. The experiments were independently repeated three times and protein bands were quantified with ImageJ (NIH).

2.12. Immunofluorescence microscopy

Bone marrow macrophages were plated on a 96‐well plate and cultured as described above. On day 5, cells were fixed with 3.7% formaldehyde and blocked with 2% bovine serum albumin (BSA). This was followed by overnight incubation with primary antibodies at 4 °C, and a washing step with 0.1% BSA and incubation with Alexa Fluor® conjugated secondary antibodies. F‐actin was labeled with Alexa Fluor® 488‐phalloidin and nuclei were stained by DAPI. Images were taken under 20 × objective by using the Leica DMi8 inverted fluorescence microscope and Leica Application Suite X software (version 3) (Leica, Concord, ON, Canada). Controls included elimination of primary antibodies from experiments and isotype controls for each antibody used.

2.13. RNA extraction, RT‐PCR and qPCR

Total RNA from cell cultures was extracted using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands), followed by the DNA Removal Kit (Thermo Fisher Scientific) and then quantified by the TECAN infinite F200 PRO with the NanoQuant Plate. The RT‐PCR was performed with One‐Step RT‐PCR Kit (Applied Biological Materials, Richmond, BC, Canada) on the T100 Thermal Cycler (Bio‐Rad). PCR products were analyzed by 2% agarose gel electrophoresis. Bands were detected using ChemiDoc™ Touch Imaging System (Bio‐Rad). Primers used were previously described (Al‐Jallad et al., 2006). The experiments were independently repeated twice. For qPCR, cDNA was synthesized from RNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real‐time PCR was performed on the StepOnePlus Real‐Time PCR System (Applied Biosystems). TaqMan® Fast Advanced Master Mix and primers were purchased from Applied Biosystems. Expression levels of Tgm1 (Mm00498375_m1), Tgm2 (Mm00436987_m1), F13a1 (Mm00472334_m1), Nfatc1 (Mm00479445_m1), Trap/Acp5 (Mm00475698_m1), Ctsk (Mm00484039_m1), Mmp9 (Mm00442991_m1), Dcstamp (Mm04209234_m1), and Ocstamp (Mm00512445_m1) were normalized to Gapdh (Mm99999915_g1). The experiments were independently repeated three times, each time in triplicate.

2.14. Statistical analysis

Data were analyzed with GraphPad Prism software (version 5.0). Results were presented as ± SEM (standard error of the mean) of three independent experiments done in triplicates. p values <0.05 were considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).

3. RESULTS

3.1. Inhibition of TG activity with NC9 blocks osteoclastogenesis

To begin our investigation whether and how TG activity regulates osteoclast differentiation, bone marrow macrophages were cultured with M‐CSF and RANKL for 5 days in the absence or presence of NC9, which is a peptidic, irreversible TG inhibitor bearing a warhead moiety that attacks and blocks the enzyme active site by binding to it covalently (Al‐Jallad et al., 2011). RANKL treatment induced the formation of TRAP+ osteoclasts as expected, and this induction was dose‐dependently and significantly inhibited by NC9 at 5–50 μM concentrations (Figures 1a and 1b). Counting the number of TRAP+ osteoclasts (nuclei ≥ 3) showed an initial, significant increase in osteoclast number which was followed by a drastic decrease and then a complete blockage of osteoclast formation (Figure 1c). Quantification of osteoclast size by counting the nuclei per osteoclast demonstrated that NC9 reduced the cell size in a dose‐dependent manner explaining the sudden increase in numbers of osteoclasts at lower concentrations, suggesting that TG activity inhibited osteoclast fusion (Figure 1d). To exclude the possibility that NC9 is toxic to macrophages/osteoclasts, cell survival analysis with MTT assay was performed. As shown in Supplementary Figure S1a, cells tolerated NC9 up to 50–60 μM level and no decrease in cell numbers was seen. The apparent increase in cell numbers is likely due to proliferation of macrophages which was permitted once osteoclastogenesis was inhibited.

Figure 1.

Figure 1

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Inhibition of TG activity with NC9 blocks osteoclastogenesis. (a) The effect of NC9 on osteoclast differentiation. Bone marrow macrophages were treated with M‐CSF and RANKL as well as different concentrations of NC9 for 5 days followed by TRAP staining at end point. Magnification bar represents 500 µm. (b) TRAP activity in the culture supernatant is reduced significantly by NC9 in a dose‐dependent manner. (c) The number of TRAP+ multinucleated (≥3 nuclei) osteoclasts is first increased followed by a dramatic decrease and complete blockage of osteoclastogenesis. (d) Number of nuclei (visualized by DAPI staining) per osteoclast in the presence of NC9 shows dose‐dependent decrease in osteoclast size. Error bars represent SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001

3.2. NC9 and other TG inhibitors T101, Z006, and MDC inhibit both differentiation and fusion of pre‐osteoclasts

Osteoclastogenesis can be divided into two steps. Monocyte/macrophage lineage precursor cells first differentiate into TRAP+ mononuclear pre‐osteoclasts which is followed by their fusion and further differentiation into multinucleated osteoclasts (Figure 2a). In our 5‐day culture system, these two stages correspond to the first 2 days and the last 3 days respectively (Supplementary Figure S2). Thus, to examine at which stage NC9 suppresses osteoclastogenesis, cells were exposed to NC9 either for the first 2 days (Exp 1) or for the last 3 days (Exp 2) (Figure 2a). As shown in Figures 2b and 2c, the number of TRAP+ mononuclear pre‐osteoclasts was significantly decreased by NC9 treatment for the first 2 days. Furthermore, the size of multinucleated osteoclasts was significantly reduced by NC9 for the last 3 days. Taken together, our results suggested that, indeed, NC9 inhibited both differentiation and fusion of pre‐osteoclasts. This was confirmed via analysis of the expression of the major osteoclast differentiation markers (Nfatc1, Trap, Ctsk, Mmp‐9) and fusion markers (Dcstamp, Ocstamp) under same experimental conditions (Exp 1 and Exp 2) in Figure 2a. Data presented in Figure 3a–f showed that NC9 in Exp 1 strongly reduced the expression of all the differentiation and fusion markers, while NC9 in Exp 2 only reduced the expression of Dcstamp.

Figure 2.

Figure 2

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TG inhibition blocks pre‐osteoclast differentiation and fusion. (a) Schematic diagram of the two phases of osteoclastogenesis, differentiation and fusion, and the experimental design for the NC9 treatments to dissect out these two functions. Exp 1 (Bone marrow macrophages treated with M‐CSF and RANKL with 20 µM NC9 for the first 2 days) examines the effect of NC9 on the differentiation of bone marrow macrophages into pre‐osteoclasts. Exp 2 (Pre‐osteoclasts treated with M‐CSF and RANKL with 20 μM NC9 for the last 3 days) examines the effect of NC9 on the fusion of pre‐osteoclasts. (b) TRAP staining of osteoclasts formed with NC9 treatment in Exp 1 and Exp 2. NC9 blocks both differentiation and fusion of pre‐osteoclasts. Magnification bar represents 500 µm. (c) Quantification of TRAP+ osteoclasts in Exp 1 and Exp 2 and number of nuclei per osteoclast in Exp 2 showing that NC9 significantly inhibits differentiation and fusion of pre‐osteoclasts. In Exp 1, the number of pre‐osteoclasts is significantly decreased. In Exp 2, osteoclast number remains the same and only size is significantly affected. Error bars represent SEM. n = 3. ***p < 0.001. NS, not significant

Figure 3.

Figure 3

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TG inhibition blocks expression of differentiation and fusion markers during osteoclastogenesis. qPCR analysis of osteoclast differentiation and fusion markers using the same treatments in Exp 1 (D0‐2) and Exp 2 (D2‐6) depicted in Figure 2a. (a) Nfatc1 (NFATc1), (b) Trap (TRAP), (c) Ctsk (CTSK), and (d) Mmp9 (MMP‐9) expression were significantly decreased during early differentiation of bone marrow macrophages to pre‐osteoclasts. Analysis of expression of fusion markers showed (e) significant Dcstamp (DC‐STAMP) decrease in both differentiation and fusion stages, and (f) significant Ocstamp (OC‐STAMP) decrease only in early differentiation phase. All the gene expressions were normalized to Gapdh. Error bars represent SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significant

To further provide evidence of TG involvement in osteoclastogenesis, we examined whether other types of TG activity inhibitors could give rise to the same results. Monodansyl cadaverine (MDC) is a dansyl containing primary amine that competes with lysine side chain in crosslinking reaction and integrates to proteins upon TG activity and thus blocks protein polymerization. Z006, a “DON” compound, is an irreversible and specific blocker of TG2 (Wilhelmus, de Jager, Smit, van der Loo, & Drukarch, 2016). T101, a 2‐[(2‐oxopropyl)thio] imidazolium derivate, is a reversible blocker of TG2 and FXIII‐A (Aleman et al., 2014; van den Akker et al., 2011). As seen in Figure 4, all the three inhibitors blocked osteoclast differentiation as evidenced by complete absence of TRAP+ cells and the undetectable TRAP activity in the culture supernatant (Figure 4a–c). As evident from Supplementary Figure S1b–d, cells tolerated all these inhibitors to the maximum concentrations tested.

Figure 4.

Figure 4

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TG inhibitors MDC, Z006 and T101 block osteoclastogenesis. TG inhibitors monodansyl cadaverine (MDC) (competitive inhibitor for protein crosslinking), Z006 (TG2 inhibitor) and T101 (TG2 and FXIII‐A inhibitor) were tested on osteoclast differentiation. Bone marrow macrophages were treated with M‐CSF and RANKL with different concentrations of inhibitors for 5 days followed by TRAP staining and colorimetric TRAP quantification in the culture supernatant. MDC (a), Z006 (b), and T101 (c) were able to inhibit osteoclast fusion at lower concentrations and block osteoclast differentiation completely at higher concentrations. Magnification bar represents 500 µm. Error bars represent SEM. n = 3. *p < 0.05, ***p < 0.001

3.3. Inhibition of TG activity with NC9 suppresses osteoclastic resorption

To measure the effect of NC9 on osteoclast function, resorption activity of mature osteoclasts was assessed. Mature osteoclasts (treated with M‐CSF and RANKL for 5 days) were formed on a bone‐like mineral surface and treated with M‐CSF and RANKL for another 5 days in the absence or presence of NC9. During this time, osteoclasts migrate and resorb the bone‐like surface which can be visualized and quantified via von Kossa staining. As shown in Figures 5a and 5b, osteoclast resorption was significantly and dose‐dependently reduced by NC9. TRAP staining during osteoclastic resorption assay confirmed the presence of osteoclasts (Supplementary Figure S3).

Figure 5.

Figure 5

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TG activity regulates osteoclast resorption. (a) Bone marrow macrophages were treated with M‐CSF and RANKL for 5 days, followed by addition of different concentrations of NC9 together with M‐CSF and RANKL for another 5 days. The resorption pits (white) were visualized with von Kossa staining and photographed. Magnification bar represents 1 mm. (b) Percentages of surface resorbed by osteoclasts at various NC9 concentrations were quantified with NIH ImageJ software. Results showed osteoclast resorption was visibly and significantly and dose‐dependently reduced by NC9. Error bars represent SEM. n = 3. **p < 0.01

3.4. TG activity is required for pre‐osteoclast migration

TGs, particularly TG2 has been linked to cell migration in previous reports (Akimov & Belkin, 2001; Cordella et al., 2017; Monteagudo, Ji, Akbar, Keillor, & Johnson, 2017; Nurminskaya & Belkin, 2012). As the migration of pre‐osteoclasts (toward other cells) is necessary for their fusion as well as for bone resorption process (to move along the surface), the effect of NC9 on the migration of pre‐osteoclasts was investigated using a cell exclusion zone assay. Pre‐osteoclasts were formed in a well with a cell seeding stopper to generate a central exclusion zone after which cells were treated with M‐CSF and RANKL in the presence or absence of 20 µM NC9 for 12 hr. Analysis of the numbers of migrated cells into the exclusion zone during this time showed that NC9 inhibited the process in a significant manner (Figures 6a and 6b), demonstrating that TG activity is important for pre‐osteoclast migration. The end point of 12 hr was chosen to eliminate potential increase in cell numbers due to proliferation as cell division generally takes 24 hr (Bernard & Herzel, 2006). Thus the cells found in exclusion zone at 12 hr time point can only arise from migration.

Figure 6.

Figure 6

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TG inhibitor NC9 inhibits pre‐osteoclast migration. (a) Pre‐osteoclast migration was investigated using a cell exclusion zone assay. Pre‐osteoclasts were formed in a well with a cell seeding stopper to generate a central exclusion zone after which cells were treated with M‐CSF and RANKL in the presence or absence of 20 µM NC9 for 12 hr. Plates were photographed at 0 and 12 hr time points. Magnification bar represents 1100 µm. (b) The number of cells migrated to the exclusion zone during the 12 hr was calculated. Results showed that NC9 inhibited the pre‐osteoclast migration in a significant manner. Error bars represent SEM. n = 3. ***p < 0.001

3.5. TG inhibitor NC9 increases RhoA levels, decreases podosome belt formation − the inhibitory effect is completely reversed with a Rho‐family inhibitor

As shown in Figure 1, NC9 induced a strong retraction and thickening of osteoclasts as evidenced by the darker staining of TRAP. Previous studies have shown that RhoA activation promotes cell retraction whereas RhoA inhibition allows cell spreading and flattening in macrophage‐derived multinucleated giant cells (Ory, Munari‐Silem, Fort, & Jurdic, 2000). Therefore, RhoA activity was investigated in NC9 treated osteoclasts. As shown in Figure 7a–c upon inhibition with NC9 for 2 days, pre‐osteoclasts showed significantly increased total RhoA levels as well elevated (albeit not significant) RhoA activity compared to the control group, suggesting that NC9 may mediate its effects via RhoA in osteoclasts. It has been shown that podosome belt forms under low RhoA activity, that is, podosome belt formation is accelerated when RhoA is inhibited (Destaing et al., 2005). To examine this, cells were stained for actin to visualize podosome belts. As seen in Supplementary Figure S4a, cells lost their podosome belts at the periphery when treated with NC9 and only actin clusters were found. To gain further evidence that increased RhoA level/activity is responsible for NC9 effects, we attempted to rescue NC9‐mediated osteoclastogenesis defect with a Rho‐family inhibitor, Exoenzyme C3. Adding 5 ng/ml Exoenzyme C3 to 10 μM NC9 treatment normalized the number and size of osteoclasts as well as rescued the podosome belt formation defect (Figures 7d and 7e). In normal, control cultures, 75% of the osteoclasts exhibited characteristic podosome belts; 10 µM NC9 treatment significantly reduced this ratio to 8%, while 5 ng/ml Exoenzyme C3 brought this back to 67% (Figures 7d and 7e). Taken together, our data demonstrate that TG activity plays an important role in regulation of RhoA and actin dynamics in osteoclast.

Figure 7.

Figure 7

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TG inhibitor NC9 increases RhoA levels, decreases podosome belt formation and the inhibitory effect is reversed with a Rho‐family inhibitor. (a) Bone marrow macrophages were treated with M‐CSF and RANKL in the presence or absence of 20 µM NC9 for 2 days. Western blotting analysis showed that NC9 increased total RhoA levels. (b) Quantification of protein bands showed NC9 significantly increased total RhoA level. The band densities were normalized to the actin loading control. Error bars represent SEM. n = 3. *p < 0.05. (c) The GTP‐bound RhoA was pulled down from whole‐cell extracts using Rhotekin RBD beads and analyzed by western blotting (not shown). Quantification of protein bands showed elevated RhoA activity, however, this was not statistically significant. Error bars represent SEM. n = 3, NS, not significant. (d) Bone marrow macrophages were treated with M‐CSF and RANKL in the presence or absence of 10 µM NC9 and 5 ng/ml Rho‐family inhibitor Exoenzyme C3 for 5 days, followed by TRAP staining (Magnification bar represents 500 µm), F‐actin and DAPI staining (Magnification bar represents 400 µm). (e) Quantification of number of osteoclasts, size of osteoclasts (number of nuclei per osteoclast) and percentage of osteoclasts with podosome belts. Adding Rho‐family inhibitor together with NC9 completely normalized the size of osteoclasts, osteoclastogenesis and podosome belt formation. Error bars represent SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significant

3.6. TG activity does not affect microtubule architecture or dynamics in osteoclasts

Studies have shown that microtubules regulate the expansion of podosome clusters into a belt and that thus disruption of the microtubule network can result in the loss of podosome belt (Destaing et al., 2005; Destaing, Saltel, Geminard, Jurdic, & Bard, 2003). Furthermore, previous studies showed that Exoenzyme C3 increased the acetylated α‐tubulin level in osteoclasts (Destaing et al., 2005), and our group has shown that NC9 regulates detyrosinated Glu‐tubulin in osteoblasts (Al‐Jallad et al., 2011; Wang, Cui, Hitomi, & Kaartinen, 2014). Examination of microtubule architecture in osteoclast after NC9 treatment for 24 hr, however, showed no major alterations (Supplementary Figure S4b) and western blotting analysis confirmed that NC9 and Exoenzyme C3 had no effect on the acetylation or detyrosination of α‐tubulin in osteoclasts (Supplementary Figure S4c).

3.7. Expression of TG1, TG2, and FXIII‐A by osteoclasts and their localization to podosome belt

Next, we were interested in understanding the levels of TG activity during osteoclastogenesis and which TG enzyme(s) are expressed and potentially involved in this process. To measure TG activity and screen TG (Tgm) family member gene expression, we collected cell lysates and mRNA from bone marrow macrophages (day 0), macrophages (day 6), and osteoclasts (day 6). As seen in Figure 8, TG activity was present in bone marrow macrophages and dramatically increased after 6 days of M‐CSF treatment, and then decreased with joint treatment with M‐CSF and RANKL. RT‐PCR analyses of TG family members: Tgm1, Tgm2, Tgm3, Tgm5, Tgm6, Tgm7, and F13a1 (band 4.2 and Tgm4 were not screened because they are exclusively expressed in red blood cells and prostate, respectively [Eckert et al., 2014]), showed clear presence of only Tgm1, Tgm2, and F13a1 genes (Supplementary Figure S5). Quantification of their mRNA expression with qPCR analysis revealed that Tgm2 mRNA was expressed at fairly high levels which remained unaltered upon differentiation cues (Figure 9a). F13a1 was significantly upregulated upon M‐CSF treatment, but significantly and dramatically reduced with joint M‐CSF/RANKL treatment (Figure 9a). Tgm1 was significantly and dramatically upregulated by M‐CSF and downregulated when RANKL was added (Figure 9a). The cellular localization of TG1, TG2, and FXIII‐A proteins were assessed by immunofluorescence microscopy in osteoclasts. All three enzymes demonstrated a similar distribution pattern: presence in the nuclear region and in the peripheral podosome belt (Figure 9b). Co‐staining of TGs with F‐actin confirmed that all the three enzymes co‐localized to podosomes (Figure 9c). Isotype control staining for TG1, TG2 and FXIII‐A antibodies were negative (Supplementary Figure S6).

Figure 8.

Figure 8

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In vitro TG activity in bone marrow macrophages and osteoclasts. TG activity in cell lysates was assessed via primary amine, 5‐(biotinamido)pentylamine (bPA) incorporation to a TG substrate protein plasma fibronectin (pFN) in a 2‐hr incubation. This was followed by detection of biotin in pFN by western blotting. Cell lysates were prepared from bone marrow macrophages (Day 0), macrophages (Day 6, M‐CSF treatment) and osteoclasts (Day 6, M‐CSF + RANKL treatment). Assay included negative controls where bPA or pFN or cell lysates were excluded (first six lanes). TG activity is seen in last three lanes. It is detected in bone marrow macrophages and dramatically increased after 6 days of M‐CSF treatment and then decreased with joint treatment with M‐CSF and RANKL

Figure 9.

Figure 9

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TG enzyme expression in bone marrow macrophages and osteoclasts and their localization in osteoclasts. (a) qPCR analysis of TG2, FXIII‐A and TG1 isoforms in bone marrow macrophages (Day 0), macrophages (Day 6, M‐CSF treatment) and osteoclasts (Day 6, M‐CSF + RANKL treatment). Tgm2 mRNA was expressed at fairly high levels which remained unaltered upon differentiation cues. F13a1 was significantly upregulated upon M‐CSF treatment, but significantly and dramatically reduced with M‐CSF and RANKL treatment. Tgm1 was significantly upregulated by both of the differentiation cues, but the upregulation induced by M‐CSF alone was significantly higher than the joint treatment of M‐CSF and RANKL. All the gene expression were normalized to Gapdh. Error bars represent SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not significant. (b) Bone marrow macrophages were treated with M‐CSF and RANKL for 5 days. TG1, TG2 and FXIII‐A in osteoclasts were visualized with their primary antibodies and Alexa Fluor® 568 (red). Nuclei were stained by DAPI (blue). TG2, FXIII‐A and TG1 (red) are found in the edge/periphery of osteoclasts. Magnification bar represents 200 µm. (c) F‐actin was visualized with Alexa Fluor® 488 (green). TG2, FXIII‐A, and TG1 (red) co‐localize with actin (green) (merge in yellow) and are all found in podosomes (arrows). Magnification bar represents 20 µm

In summary, our work confirms the new role for TGs and TG activity in osteoclastogenesis. TG activity is needed for differentiation, migration and fusion of osteoclasts and regulates actin dynamics in these cells.

DISCUSSION

Previous studies, including ours, have linked TG enzymes and their activity to monocyte, macrophage, and osteoclast function in vitro and in vivo (Adany et al., 2001; Akimov & Belkin, 2001; Bagoly et al., 2012; Chrobok et al., 2017; Kim et al., 2017; Mousa et al., 2017; Raghu et al., 2015) and our recent work demonstrated the relevance of TG2, FXIII‐A in regulation of osteoclastogenesis in mice as well as showed for the first time the presence of TG1 in osteoclasts (Mousa et al., 2017). In the present study, we have continued the exploration on the role of TGs in osteoclastogenesis and show that TG inhibitors are highly potent blockers of osteoclast differentiation, fusion and migration. These functions for TG activity are aligned with several previous findings on the involvement of TGs in signaling pathways related to osteoclastogenesis, cell fusion, and migration.

Inhibition of TGs/TG activity by NC9 blocked macrophage differentiation to osteoclasts. Analysis of osteoclast markers showed that TG inhibitor significantly downregulated Nfatc1, Trap, Cstk, and Mmp9 in the first two days of treatment with M‐CSF and RANKL which induces differentiation from bone marrow macrophages to pre‐osteoclasts. NC9 did not have significant effect on these genes after the initial differentiation occurred, that is, fusion stage between day 3 and 6. Pre‐osteoclast fusion to multinucleated mature osteoclasts is regulated by master fusogenic factors Dcstamp and Ocstamp (Witwicka et al., 2015; Yagi et al., 2005; Yang et al., 2008; Zhang et al., 2014) which were both significantly downregulated by NC9 in the first two days of treatment with M‐CSF and RANKL. As it is well known that NFATc1 is the master regulator of osteoclast differentiation and transcriptionally regulates Trap (Takayanagi et al., 2002), Ctsk (Matsumoto et al., 2004), Mmp9 (Sundaram et al., 2007), Dcstamp and Ocstamp (Kim et al., 2008; Miyamoto et al., 2012), the inhibited expression of Nfatc1 may be responsible for the decreased expression of the others. The difference in the expression levels of the markers in NC9 non‐treated cells reflects the different stages of the cells in Exp 1 (end point at day 2) and Exp 2 (end point day 6) as illustrated in Figure 2a. Number of studies showed similar gene expression patterns at early and late differentiation stages (Hoshino et al., 2013; Kogawa et al., 2010; Kopesky et al., 2014). The observation that NC9 downregulated Nfatc1 seemingly contradicts to the work of Kim et al. (2017) which showed that TG2 knockdown led to elevated Nfatc1/NFATc1, at both mRNA and protein level, and enhanced NFATc1 transcriptional activity in osteoclasts. However, in their study, except for the weakly detected FXIII‐A mRNA, TG2 was the only TG family member expressed at a high level in macrophages and pre‐osteoclasts, whereas our study showed clear presence of TG1, TG2 and FXIII‐A during differentiation and more importantly, the induction of TG1 and FXIII‐A with M‐CSF. Furthermore, their study did not examine the effect of TG2 knockdown on the expression of other TGs or TG activity in general.

Our work shows that TG activity is required for pre‐osteoclast migration. TGs, particularly TG2 has been linked to cell migration in several previous publications (Akimov & Belkin, 2001; Cordella et al., 2017; Monteagudo et al., 2017; Nurminskaya & Belkin, 2012). Akimov et al. showed cell surface TG2 acted as an integrin‐associated adhesion receptor involved in extravasation and migration of monocytes into tissues (Akimov & Belkin, 2001). Monteagudo et al. (2017) showed that ablation of TG2 significantly inhibited migration of primary astrocytes and this migration defect was only rescued with the native protein, not with mutants lacking activity strongly suggesting that TG activity of TG2 is driving migration (versus other non‐enzymatic functions of TG2). It was also reported that TG2 can affect cell migration through crosslinking PDGF‐BB (Cordella et al., 2017), which interestingly is a highly potent chemotactic factor known to promote monocyte migration (Bethel‐Brown, Yao, Hu, & Buch, 2012; Cho et al., 2016; Siegbahn, Hammacher, Westermark, & Heldin, 1990). However, it is likely that the effects we see in our work arise from TG2 function in podosomes where it localizes.

In our work we show that TG inhibition by NC9 increases RhoA levels in cells and inhibits podosome belt formation in osteoclasts. Inhibitor of RhoA‐family, Exoenzyme C3, reverses all effects of NC9 strongly suggesting that mechanism of action of TGs in osteoclastogenesis involved Rho GTPases. RhoA, one of the best studied members of Rho GTPases, has been found to control podosome patterning in osteoclast (Ory, Brazier, Pawlak, & Blangy, 2008). Osteoclasts on mineralized surfaces form a sealing zone that allows isolated resorption to occur, however, when osteoclasts are seeded on glass surfaces they do not form a sealing zone, but a podosome belt as in our study (Jurdic et al., 2006). The requirement for RhoA activity was shown to be different for the two surface phenotypes (sealing zone versus podosome belt) of osteoclasts, that is, high basal RhoA activity was required for sealing zone formation (Saltel, Destaing, Bard, Eichert, & Jurdic, 2004) but it did not trigger sealing zone formation in osteoclasts seeded on glass (Destaing et al., 2005; Ory et al., 2000). On the other hand, RhoA inhibition accelerated podosome belt formation in osteoclasts seeded on glass (Destaing et al., 2005), but disrupted the sealing zone and triggered the podosome belt formation in osteoclasts seeded on bone (Saltel et al., 2004), indicating that lowering the RhoA activity in cells is required for podosome belt formation. In addition, a previous study showed that upregulated expression of RhoA suppressed osteoclastogenesis and treatment with the Rho‐family inhibitor (Exoenzyme C3) rescued the impaired osteoclastogenesis (Mizoguchi, Murakami, Saito, Miyasaka, & Kohsaka, 2013). In this study, we show that inhibition of TGs increases RhoA levels in cells. Furthermore, we show here that TG1, TG2 and FXIII‐A co‐localize to the podosomes where RhoA is also found in osteoclasts (Jurdic et al., 2006). Although the precise mechanism by which TGs may regulate RhoA activity (or other Rho‐family members) in osteoclasts is likely complex and remains to be explored, previous findings have shown several links of TGs to RhoA. It was shown that TG2 can promote RhoA activation through integrin clustering and inhibition of the Src‐p190RhoGAP signaling pathway in fibroblasts (Janiak, Zemskov, & Belkin, 2006). Also, Guilluy et al. have shown that TG2‐mediated serotonylation leads to RhoA activation and increased proteasomal degradation in vascular smooth muscle cells (Guilluy et al., 2007). This activation of RhoA due to TG2‐mediated serotonylation was also involved in pulmonary artery smooth muscle cell proliferation (Guilluy et al., 2009). We have also demonstrated that NC9 can target FXIII‐A in preadipocytes and affect RhoA/ROCK activity (Myneni, Hitomi, & Kaartinen, 2014). No data are available on TG1 and Rho GTPases. While these findings where TG2 is shown to activate RhoA might seem contradictory to our current study, there are also several studies showing that TG2 can increase the levels of active Rac1 (through inhibiting the Bcr GTPase‐activating activity in COS‐1 cells [Yi, Groffen, & Heisterkamp, 2009]) which is known to antagonize RhoA function in osteoclasts (Ory et al., 2000). Therefore, TG2 may promote Rac1 activity and inhibit RhoA activity in osteoclasts. However, all above data on TG2 and RhoA can only very carefully be extrapolated to our work where three TGs appear involved in the process and other Rho‐family members might contribute to the effects. More work need to be done to explore which TGs and how they control RhoA or Rho‐family activity in macrophages and osteoclasts. This is the focus of our future work.

Our previous study demonstrated that the TG enzymes expressed in osteoclasts are TG2, FXIII‐A and TG1 and here we also confirm their mRNA expression and cellular localization and show their differential expression patterns during osteoclastogenesis. Our data show that TG2 is expressed at similar levels in bone marrow macrophages (day 0), macrophages (day 6), and osteoclasts (day 6) with no apparent upregulation, whereas FXIII‐A is expressed at high levels in bone marrow macrophages (day 0) and macrophages (day 6), but downregulated dramatically in osteoclasts (day 6). TG1 in turn shows a significant increase with M‐CSF treatment in macrophages (day 6) and with M‐CSF and RANKL treatment in osteoclasts (day 6), but the upregulation by M‐CSF alone is higher than the M‐CSF/RANKL treatment. These different expression patterns suggest that the three TGs may play distinct roles in osteoclastogenesis. In our study, we found that inhibitors NC9 and MDC were more potent than inhibitors Z006 (inhibits TG2) and T101 (inhibits TG2 and FXIII‐A). Collectively, the inhibitor data may suggest the following: 1) Z006 and T101 may target specific TGs at lower concentrations but inhibit all the three TGs at higher concentrations; 2) mechanism of action likely involves protein crosslinking (in addition to potentially involving deamination reaction or non‐enzymatic functions of TGs [Iismaa et al., 2009; Kanchan, Fuxreiter, & Fesus, 2015]) because MDC, competitive inhibitor of protein crosslinking, was able to block osteoclastogenesis. Our future studies will include dissecting out these functions. The discovery of TG1 in macrophages and osteoclasts in our previous work and in this study suggests that it has a role in bone remodeling. TG1, also named as keratinocyte TG, is responsible for the formation of cornified cell envelope that acts as protective skin barrier (Eckert et al., 2014). Mutations in TGM1 causes lamellar ichthyosis in humans, an autosomal recessive skin disorder (Huber et al., 1995; Parmentier et al., 1995; Russell et al., 1995). Tgm1/TG1 expression has not been previously documented in any bone cells prior to our work (Mousa et al., 2017) and no skeletal phenotype has been reported for Tgm1 deficient mice which die within 4–5 hr after birth (Matsuki et al., 1998).

In conclusion, our study describes a role for TGs in osteoclastogenesis. Inhibitors of TGs may provide excellent anti‐resorptives that could target all stages of osteoclastogenesis.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Supporting information

Additional Supporting Information may be found online in the supporting information tab for this article.

Fig. S1. The effect of TG inhibitors, NC9, MDC, Z006 and T101 on cell viability in osteoclasts.

Fig. S2. TRAP staining images of different stages of osteoclasts and their precursors.

Fig. S3. TRAP staining images of osteoclasts during osteoclastic resorption assay.

Fig. S4. The effect of NC9 on podosome belt formation and microtubule architecture and dynamics in osteoclasts.

Fig. S5. RT‐PCR analysis of TG family members bone marrow macrophages (D0), macrophages (D6) and osteoclasts (Day 6).

Fig. S6. The isotype controls for antibodies against TG1, TG2 and FXIII‐A used in FIGURE 9.

JCP-233-7497-s001.pdf (566.4KB, pdf)

ACKNOWLEDGMENTS

The authors thank Aisha Mousa for helpful assistance and Dr. Svetlana Komarova (Faculty of Dentistry, McGill University) for helping with osteoclast cultures. This study was supported by grants to MTK from the Canadian Institutes of Health Research (CIHR) (MOP‐119403). HS is supported by stipend from the Faculty of Dentistry of McGill University. MTK is members of the Fonds de Recherche—Santé (FRQ‐S) Network for Oral and Bone Health Research.

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Associated Data

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Supplementary Materials

Additional Supporting Information may be found online in the supporting information tab for this article.

Fig. S1. The effect of TG inhibitors, NC9, MDC, Z006 and T101 on cell viability in osteoclasts.

Fig. S2. TRAP staining images of different stages of osteoclasts and their precursors.

Fig. S3. TRAP staining images of osteoclasts during osteoclastic resorption assay.

Fig. S4. The effect of NC9 on podosome belt formation and microtubule architecture and dynamics in osteoclasts.

Fig. S5. RT‐PCR analysis of TG family members bone marrow macrophages (D0), macrophages (D6) and osteoclasts (Day 6).

Fig. S6. The isotype controls for antibodies against TG1, TG2 and FXIII‐A used in FIGURE 9.

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