Undercarboxylated osteocalcin inhibits the early differentiation of osteoclast mediated by Gprc6a

Cells cultures

All the cell lines used in the present study were obtained from Chinese National Infrastructure of Cell Line Resource. RAW264.7 cells were cultured in DMEM (HyClone, USA) supplemented with 10% FBS (Gibco, USA) and incubated at 37 °C in 5% CO₂ air humidified incubator. BMMs were isolated from the long bones of 6-week-old mice and maintained in α-minimal essential medium containing 10% FBS in the presence of MCSF (10 ng/ml, Pepro Tech., USA). To generate osteoclasts from BMMs, cells were plated in 24-well tissue culture plates and cultured in the presence of 30 ng/ml MCSF and 50 ng/ml RANKL (R&D System, USA).

The ucOCN production

GST-OCN fusion protein was bacterially produced and purified by Glutathione Sepharose Fast Flow column (GE Healthy Life Science, USA) according to standard procedures. OCN protein/gene was then cleaved out from the GST subunit using thrombin. Its purity and concentration were evaluated by ELISA (Clontech, USA).

Cell migration assays

Migration was measured by wound healing assay. RAW264.7 cells were plated at a density of 4 × 10⁵ cells/ml into 24-well plates. When cells achieved 80% confluence, the plates were streaked with a sterile micropipette tip. After rinsing off the released cells, the plates were visualized at 10 × magnification along with the streaks at time 0 and 24 h later. The cell migration was determined by measuring wound width (in pixels) using ImageJ software (NIH, USA).

Cell proliferation analysis by MTS

RAW264.7 cells were seeded into a 96-well plate at a density of 1 × 10⁵ cells/well, and replaced medium with different concentration of ucOCN (1, 10, 100, 1,000 ng/ml) after adhesion about 24 h. Then, the effect of different concentrations of ucOCN on RAW264.7 cell proliferation was analyzed using the MTS assay kit according to the instructions of the manufacturer (Promega, USA).

RT-qPCR for gene expression analysis

Total RNAs were extracted using the Trizol reagent according to the standard protocol. The cDNA was synthesized from 500 ng of total RNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian China) according to the manufacturer’s instructions, and performed the analysis of gene expression using TB Green™ Ex Taq™ II (Tli RnaseH) from Takara on a Roche lightCycler 96 instrument with 0.4 µM gene-specific primer pairs (Table 1) respectively; GAPDH amplification was used as an internal reference for each sample.

TRAP staining

RAW264.7 cells were seeded in a 6-well plate with 100 ng/ml ucOCN in the presence of 50 ng/ml RANKL. BMMs were treated with ucOCN (100 ng/ml) in the presence of 30 ng/ml MCSF and 50 ng/ml RANKL. The medium was replaced every alternate day. After 4 or 6 days of culture, TRAP histochemical staining was performed using a leukocyte acid phosphatase kit (Sigma,USA) following the instruction.

Toluidine blue staining of bovine bone slices for osteoclast resorption

Toluidine blue was a basic thiazine metachromatic dye with high affinity for acidic tissue components and widely utilized for bone resorption pit staining. In brief, bovine slices (Beijing Keruimei technology CO. LTD) were dipped in PBS and medium with 10-fold high concentration of streptomycin and penicillin for 12 h, respectively. RAW264.7 cells or BMMs were seeded onto the slices at a density of 1 × 10⁵ cells/well with the inducing factors or coupled with ucOCN treatment. The medium was replaced every alternate day. After 10 days of culture, the slices were washed with PBS followed by fixation with 4% paraformaldehyde for 20 min. After this, cells were washed twice with PBS and removed from bone slices via ultrasonication in 0.25 M NH₄OH for 3 times followed by distilled water. Resorption pit formation was stained by 0.1% toluidine blue (Amersco, USA) for 10 min at room temperature. The slices were then rinsed with distilled water more than 5 times to excluderesidues. Resorption pits are now stained in dark blue and images were taken via light microscopy.

Actin ring staining

Actin rings of mature osteoclasts were detected by actin filaments staining with rhodamine-conjugated phalloidin (molecular probe, USA). Mature osteoclasts were formed from BMMs cultures in the presence of RANKL (50 ng/ml) and MSCF (30ng/ml). At the end of incubation with ucOCN treatment for 6 days, osteoclasts were stained with rhodamine-conjugated phalloidin for actin. The distribution of actin rings was visualized and the images were taken by a Nikon Eclipse Ti microscope (Japan).

Western blot analysis

The cells were lysed by RIPA buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, and 0.1% sodium dodecyl sulfate) containing protease inhibitor cocktail (Roche, German). Each sample was kept on ice for 30 min before it was centrifuged at 13,000 g for 30 min at 4 °C. The protein concentration of the supernatant was determined with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, USA). The total proteins were subjected to SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h and then incubated overnight with a primary antibody diluted in 5% bovine serum albumin at 4 °C (specific antibodies for RANK, c-Fms (Abcam, UK), P44/42 MAPK(REK1/2), Phospho-p44/42 MAPK(ERK1/2), GAPDH (CST, USA), Grpc6a (Santa Cruz, USA)). The membranes were incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (CST, USA) for 2 h at room temperature. Signals were detected using enhanced luminescence (Bio-Rad, USA) after washing three times. The intensity of the protein bands was calculated using ImageJ software (NIH, USA).

Luciferase reporter gene assay

RAW264.7 cells were seeded into 24-well plates at a density of with 1.5 × 10⁵ cells/ml and cultured for 12 h. The specific siRNA of Gprc6a (siRNA-2553 GCAGAAGACTAACACCAAA) was transfected into RAW264.7 cells for 24 h before reporter transfection and ucOCN treatment. The reporter plasmid pGL4.33[luc2P/SRE/Hygro] or pGL4.29 [luc2P/CRE/ Hygro] coupled with phRLTK was transfected into RAW264.7 cells followed by treatment with 100 ng/ml ucOCN, 20 mM L-ornithine (Sigma, USA) alone or combined with 30 µM NPS2143 (Sigma, USA) as previous reports desrption (Rueda et al., 2016) 36 h later. The measurement of luciferase activity was performed with the Dual Luciferase Reporter Assay kit (Promega, USA) following the manufactory’s instruction.

Statistical analysis

Experiments were carried out independently at least three times. Results are expressed as the mean ± SEM and were compared by Student’s t test (two groups) or Two-way repearted measures ANOVA with Duncan’s multiple comparison (multiple groups). Results were considered significantly different for p < 0.05.

ucOCN attenuated the proliferation and migration of RAW264.7 cells

First of all, as the previous report (Oury et al., 2011), we purified ucOCN from an artificial E.coli prokaryotic bacteria by GST-fusion method. Its purity and concentration were examined by specific ELISA kit. As shown in Fig. S1, no cOCN was detected by specific ELISA kit and a very low of endotoxin level was detected. The purified ucOCN was used in the following experiments.

To explore the effects of ucOCN on osteoclast proliferation, the mouse monocyte RAW264.7 cells were treated with different concentration of ucOCN (1, 10, 100, 1,000 ng/ml) for 24 h, 48 h and 72 h. The growth rate of the different treated cells was detected by MTS method and it was significantly inhibited by ucOCN treatment at the indicated time point as shown in Fig. 1A. The inhibition effect of ucOCN at the lowest concentration (1 ng/ml) just lasted for 48 h, but the inhibition effects of ucOCN at other three concentrations (10, 100, 1,000 ng/ml) still significantly occurred at 72 h time point. There was about 40% inhibition at 24 h and 48 h, but only 20–30% at 72 h. These results indicated that ucOCN attenuated the proliferation activity of RAW264.7 cells with concentration and time-dependant pattern.

The migration of RAW264.7 cells was measured in a scrape wounding assay over 24 h with ucOCN (100 ng/ml) treatment or not. As shown in Figs. 1B–1C, there was a higher motility in untreated RAW264.7 cells than ucOCN treated cells. In the control group, the cells at the edge of the steak were significantly migrated toward the middle and the width of the scribe line became narrowed. But there were almost no changes in the ucOCN treated group. The above results suggested that ucOCN inhibited the motility of RAW264.7 cells.

ucOCN suppressed RANKL-induced osteoclastogenesis

ucOCN affected the osteoclastic gene expression. To clarify the effects of ucOCN on osteoclastogenesis, we examined the osteoclastic gene expression during induction in the presence of ucOCN by RT-qPCR. The preosteoclastic RAW264.7 cells were induced to osteoclastic differentiation by 50 ng/ml receptor activator of nuclear factor- κ B ligand (RANKL), a key osteoclastogenic cytokine, without or with ucOCN (10 ng/ml or 100 ng/ml based on the proliferation effects results) for 4 days, then lysed by Trizol for gene expression assay. As shown in Fig. 2A, the marker genes (NFATc1, RANK, TRAF6, etc.) of osteoclastic differentiation were significantly increased following RANKL induction, especially the upregulation of CathK and TRAP genes about several hundred folds. Compared with the ucnOCN untreated control group (CN) coupled with RANKL induction, the expression of these genes dramatically decreased about 40–60% after treatment with ucOCN accompanied with RANKL induction. The inhibition efficiency of 100 ng/ml ucOCN was higher than that of 10 ng/ml (Fig. 2B) and it was reported that serum concentration of ucOCN could be 35.9 ng/mL in mice (Prats-Puig et al., 2014), So 100 ng/ml was used in the following experiments.

Secondarily, mature TRAP positive multinucleated cells (MNCs) were assayed in RAW276.4 cells and BMMs. Cells were incubated with 100 ng/mL ucOCN in the presence of RANKL for RAW264.7 cells or in the presence of RANKL and MCSF for BMMs. RAW264.7 cells differentiated to mature TRAP-positive MNCs in the absence of ucOCN, however, fewer MNCs formation was observed in the presence of 100ng/ml ucOCN (Fig. 3A). ucOCN inhibited the formation and number of TRAP positive MNCs. In BMMs, ucOCN appeared to have similar effects. Indeed, there were no purple TRAP positive cells after treatment of MCSF or combined with ucOCN, neither mononuclear cells or multinucleated cells (Fig. S2). The treatment of BMMs with RANKL and MCSF induced the formation of TRAP positive MNCs, nevertheless ucOCN decreased the induction effects of RANKL to osteoclast (Fig. 3C).

We further examined the effects of ucOCN on the function of mature osteoclasts to resorb bone. RAW264.7 cells were plated onto bone slices and induced with RANKL in the presence of 100 ng/mL ucOCN. RANKL stimulated cells form a number of pits, which indicated successful induction of RAW264.7 cells to functional bone resorption activity by RANKL. As shown in Fig. 3B, treatment of 100 ng/ml ucOCN significantly reduced the formation of resorption pits in number and areas compared with treatment with RANKL alone. Similar results were observed in BMMs after induced with MCSF+RANKL combined with ucOCN (Fig. 3D). During bone resorption, an actin ring was formed to seal the resorption area. To investigate the effects of ucOCN on actin ring formation, immunofluorescence staining was carried out for BMM cells induced by RANKL in the presence of ucOCN. There were single cells stained with red in only MCSF treatment group. Well-formed actin rings were observed in the MCSF and RANKL-treated cells. After the addition of ucOCN during the progress of induction of MCSF and RANKL, the number of red actin rings decreased, and the structure of formed actin rings showed irregularity and shrinkage (Fig. 3E). These results indicated the inhibitory effects of ucOCN on the bone resorption of osteoclast and the formation and integrity of the osteoclast actin ring.

ucOCN affected the initial differentiation of osteoclasts

The above results suggested the inhibition effects of ucOCN on osteoclast differentiation. Next, we tried to explore which stage of osteoclast differentiation was affected by ucOCN. To achieve this aim, ucOCN was added to the MCSF+RANKL inducing medium in the final concentration of 100 ng/mL for BMM cells at different time points as indicated (0, 2nd, 4th day) in Fig. 4A, then some classic osteoclastic genes were detected by RT-qPCR. MNCs were assayed by TRAP staining at the 6th day. Compared with the ucOCN untreated group (CN), the expressions of RANK, TRAP, CathK, and CTR were significantly decreased about 20–50% when ucOCN was simultaneously added with inducing reagent, and only descent tendency was observed when ucOCN was added at the 2nd day or almost no effects were detected when ucOCN was added at the 4th day (Fig. 4B). The inhibition ratio of ucOCN on the expression of classic osteoclastic genes at 0 day (20–50%) was similar to that of RAW264.7 cells as shown in Fig. 2B (40–60%). There was abundant of TRAP positive MNCs in the MCSF and RANKL inducing group, which was markedly reduced in the ucOCN additional group at day 0. The number of TRAP positive MNCs showed a slight decreased in the 2nd day group and no changes occurred in the 4th day group when compared with the ucOCN untreated inducing group (Fig. 4C). It was accordant with the results of gene expression. These results demonstrated that ucOCN could attenuated the early differentiation of osteoclasts.

To further confirmation this phenomenon, RAW264.7 cells were added with 100 ng/mL ucOCN for 2 days, then evaluated the expression of c-Fms (the receptor of MCSF) and RANK (the receptor of RANKL) genes by RT-qPCR, western blot. As shown in Figs. 4D–4F, the mRNA and protein expression of c-Fms and RANK were inhibited about 30–50% after addition of ucOCN. These results suggested that ucOCN could inhibit the expression of RANK and c-Fms in RAW264.7 cells.

Gprc6a mediated the effects of ucOCN on osteoclasts

ucOCN promoted beta-cells proliferation and testosterone synthesis in Leydig by Gprc6a, an orphan G protein-coupled receptor (Oury et al., 2011; Wei et al., 2014). Gprc6a is a cation-, calcimimetic-, and OCN-sensing receptor and widely expressed in brain and peripheral tissues, with the highest levels in kidney, skeletal muscle, testis and leucocyte (Wellendorph & Brauner-Osborne, 2004). but it was unknown if osteoclastic lineage cells expressed Gprc6a. In this regard, The expression of Gprc6a in RAW264.7 cells and BMMs were detected by RT-PCR, western blot, and immunofluorescent staining method. Similar to the positive control TC-6 cells, bands of the desired size were observed in the agarose gel electrophoresis of RT-PCR production and in the PVDF film of protein by western blot detection from both osteoclastic lineage cells (Figs. 5A and 5B). Immunofluorescent staining also demonstrated the expression of Gprc6a, which localized in the cytoplasm membrane of RAW264.7 cells and mature osteoclast (Fig. 5C).

Several reports have demonstrated that ucOCN could activate ERK and serum response element (SRE)-luciferase reporter activity (Pi et al., 2005), a transcriptional effector CREB in Leydig (Oury et al., 2011). To evaluate if Gprc6a was activated by ucOCN in osteoclastic linage cells, the SRE- or CRE- (cAMP response element) luciferase reporter was transfected into RAW264.7 cells coupled with Renilla phRLTK vector. After transfection for 36 h, the cells were treated with ucOCN, L-ornithine (an agonist of Gprc6a) with or without 10 µM NPS2143 (non-competitive antagonist of Gprc6a (Faure et al., 2009)), and then the transcriptional activity was measured with the Dual Luciferase Assay. To screen an optimal time point, the cells were treated for 6 h, 12 h, and 24 h after 36 h of reporter transfection, as illustrated in Fig. 5D. The luciferase activity was significantly increased after treatment with ucOCN at the indicated time, especially at 12 h which was performed in the following experiment (Fig. 5E). Although there was no effect of NPS2143 on the luciferase activity, the promotive effects were eliminated by NPS2143 co-treatment, which was concordance with the positive control groups of L-ornithine (Fig. 5F). Similar results were achieved by CRE-luciferase detection (Fig. 5G). Moreover, increased ERK1/2 phosphorylation was observed after 12 h treatment of ucOCN or L-ornithine, which was also wiped out by NPS2143 (Fig. 5I). Next, a Gprc6a specific siRNA (Pi et al., 2010) was transfected into RAW267.4 cells for 48 h, followed by CRE-luciferase reporter transfection and ucOCN treatment as the above description. As shown in Fig. 5H, the promotion of ucOCN and L-ornithine to CRE luciferase activity was completely ablated by the siRNA. These results implied that ucOCN promoted SRE/CRE signaling and ERK1/2 phosphorylation mediated by Gprc6a.