Hao Hu1,2, Chao Li1,2, Haitao Zhang1,2, Gang Wu1,2, Yong Huang1,2. 1. Department of Traditional Chinese Traumatology, Hubei Provincial Hospital of Traditional Chinese Medicine, Wuhan, Hubei 430061, P.R. China. 2. Department of Traditional Chinese Traumatology, Hubei Province Academy of Traditional Chinese Medicine, Wuhan, Hubei 430070, P.R. China.
Osteoclast function is important in clinical disorders of bone resorption, such as osteoarthritis, rheumatoid arthritis and osteoporosis. Vasodilator-stimulated phosphoprotein (VASP) has been demonstrated to be involved in several cell processes, including differentiation and mobility (1). VASP has been reported to serve an important role in cell migration and tumour metastasis (1). Previously, we demonstrated VASP protein regulates osteosarcoma cell migration and metastasis, potentially through interactions with Ras-related C3 botulinum toxin substrate 1 (Rac1) (2). VASP expression was significantly higher in patients with metastatic osteosarcoma compared with patients with non-metastatic osteosarcoma (2). VASP, a member of the Ena/VASP family, links the cytoskeletal system to signal transduction pathways and plays an essential role in cytoskeletal dynamics. VASP, therefore regulates cell morphology, adhesion and migration. In addition to its role in normal cell growth, embryonic development and homeostasis, VASP plays an essential role in numerous diseases, such as cancer metastasis, thrombosis, arteriosclerosis and nephritis (2-5). Notably, VASP has been shown to serve important roles in the stimulation and inhibition of cell migration (2,4). Osteoclasts are bone-resorbing cells that originate from hematopoietic precursor cells. Osteoclasts require colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL) for their survival, proliferation, differentiation and activation. The binding of RANKL to its receptor RANK triggers osteoclast precursors to differentiate into osteoclasts (6,7). Thus, in the present study, the effects of VASP in osteoclast differentiation and mobility were investigated, providing a basis for exploring novel mechanisms of bone diseases. The molecular details of the functional role of VASP in cell motility and migration remain unknown. NF-κB signalling mediates RANK ligand-induced osteoclastogenesis, and the inhibition of NF-κB has been proposed as an effective approach to inhibiting osteoclast formation and bone resorptive activity (8,9). The transcription factor NF-κB consists of several subunits capable of crossing the nuclear membrane, binding to specific promoters and regulating various signalling pathways, which are essential for normal cellular functions and development, including skeletal development (8,9). The present study investigated the effects of silencing VASP on the expression of αV-integrin and tartrate-resistant acid phosphatase (TRAP), and lamellipodia protrusion in murine macrophage RAW264.7 cells.
Materials and methods
Materials
Recombinant murinesRANKL (cat. no. 315-11C) was purchased from PeproTech, Inc. The antibodies used for fluorescent microscopy were as follows: Anti-VASP (cat. no. 13472-1-AP; 1:100; ProteinTech Group, Inc.), Phalloidin (cat. no. P5282; 1:100; Sigma-Aldrich; Merck KGaA), DAPI (1:1,000; cat. no. C1002; Beyotime Institute of Biotechnology). Antibodies used for western blotting experiments were anti-GAPDH (cat. no. 60004-1-Ig; 1:50,000; ProteinTech Group, Inc.), anti-VASP (cat. no. 13472-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-TRAP (cat. no. sc-376875; 1:1,000; Santa Cruz Biotechnology, Inc.), anti-αV integrin (cat. no. 10569-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-IκBα (cat. no. 10268-1-AP; 1:1,000; ProteinTech Group, Inc.), anti P-IκBα (cat. no. 13921-1; 1:1,000; Cayman Chemical Company), anti-NF-kB p65 (cat. no. ABP0167), anti-lamin A (cat. no. ABP0098), anti-nuclear factor of activated T cells cytoplasmic 1 (NFATc1) (cat. no. ABP53112) (all 1:1,000; Abbkine Scientific Co., Ltd.) and c-Fos (cat. no. AF6489; 1:1,000; Beyotime Institute of Biotechnology). Cy3-labeled goat anti-mouse was used as secondary antibody (cat. no. A0521; 1:500; Beyotime Institute of Biotechnology).
Cell culture and transfection
The RAW 264.7mouse monocyte macrophage cell line was used as an osteoclast precursor. RAW 264.7 cells were obtained from the American Type Culture Collection and maintained in α-MEM (cat. no. PM150421; Procell Life Science & Technology Co., Ltd.) containing 10% FBS (cat. no. 10099-141; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (cat. no. ST488; Beyotime Institute of Biotechnology) in a humidified atmosphere (5% CO2, 37˚C). Cells were checked for mycoplasma contamination on regular basis. After 3 days of cell growth, the RAW 264.7 cells were treated with 50 ng/ml sRANKL for 5 days in order to induce cell differentiation (i.e. osteoclastogenesis) (10,11). sRANKL was dissolved in PBS and PBS alone was used as the mock control.For establishing stable clones of the VASP-siRNA, scrambled and empty vector, 5x103 RAW 264.7 cells were seeded per well in 6-well plates (2). After the cells were grown to 70-80% confluence, transfection was performed using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.). After preparation of the Lipofectamine 2000/siRNA mix according to the manufacturer's protocol, 200 µl mix (5 µl Lipofectamine 2000 and 10 µl siRNA in MEM without FBS; total 200 µl) was added to each well and the cells were incubated at 37˚C in a 5% CO2 incubator for 6 h. Stable clones were selected using blasticidin (cat. no. ST018; Beyotime Institute of Biotechnology) at 5 µg/ml for 3 weeks. VASP was silenced using specific siRNA (synthesized by Guangzhou RiboBio Co., Ltd.) which are as follows: forward, 5'-GGGCUACUGUGAUGCUUUATT-3' and reverse, 3'-TTCCCGAUGACACUACGAAAU-5' and the scrambled sequence was used as a negative control (forward, 5'-UUCUCCGAACGUGUCACGUTT-3' and reverse, 3'-TTAAGAGGCUUGCACAGUGCA-5'). Prior to subsequent experiments, cell were cultured in α-MEM containing 10% FBS for 2-6 h.
Isolation of RNA and RT-PCR
Total RNA was extracted from RAW 264.7 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. First-strand complementary DNA (cDNA) was synthesized from the total RNA in a final volume of 20 µl using Superscript II reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) containing 1 µl Oligo(dT)12-18, 1 µl dNTP Mix, 4 µl 5X First-Strand Buffer and 1 µl reverse transcriptase. cDNA was amplified using gene specific primers via PCR. The PCR conditions were as follows: A holding stage of 94˚C for 3 min, followed by 40 cycles at 95˚C for 30 sec, 60˚C for 30 sec and 72˚C for 30 sec. Primers used are listed as follows: VASP forward, 5'-GGGCTACTGTGATGCTTTA-3' and reverse 5'-GAATGATGGCACAGTTGATA-3 (174 bp); αV integrin forward, 5'-TTCTCGGTGGTCCTGGTAG-3' and reverse, 5'-ACATCTGCGTAATCATCCCC-3' (370 bp); TRAP forward, 5'-CCCAGCCCTTACTACCGTTT-3' and reverse, 5'-TGCTTTTTGAGCCAGGACAG-3' (176 bp); and GAPDH forward, 5'-ATGGGTGTGAACCACGAGA-3' and reverse, 5'-CAGGGATGATGTTCTGGGCA-3' (229 bp). Relative gene expression was calculated using the standard 2-ΔΔCq method (12). Data are presented as the fold-change in gene expression normalized to the level of GAPDH.
Microscopy
Cells were seeded onto glass coverslips in 24-well plates at concentration of 5x103 cells/well in medium (α-MEM and 10% FBS) with 50 ng/ml sRANKL for 5 days with or without knockdown of VASP. Following incubation, coverslips were washed three times with PBS. Cells were fixed with 4% formaldehyde for 15 min and then washed with PBS thrice at room temperature. Before blocking with 5% BSA (cat. no. SW3015; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min cells were immersed in 0.5% Triton X-100 for 20 min and washed thrice with PBS at room temperature. VASP antibody (1:100) was added and the cells were incubated overnight at 4˚C. The next day, the cells were incubated with goat anti-mouse polyclonal secondary antibody for 1 h at room temperature. After washing the cells with PBS, Phalloidin (5 µg/ml) was added for F-actin staining and the cells were incubated for 1 h at room temperature. Subsequently, cells were washed with PBS and DAPI (5 µg/ml) was added to stain the nuclei for 5 min. Representative images were captured using a C2+ confocal microscope system (Nikon Corporation) at x400 magnification.
Trypan blue exclusion assay (viability assay)
RANKL-treated RAW264.7 cells were harvested and suspended with 0.4% trypan blue solution (cat. no. ST798; Beyotime Institute of Biotechnology) for 3 min at room temperature. The cells were counted using a haemocytometer under an inverted microscope (CKX53; Olympus Corporation) and cells that were observed to exclude the dye were considered viable, as described previously (13).
TRAP staining
TRAP staining was performed to determine the pre-osteoclasts (RAW264.7 cells) and multinuclear mature osteoclasts using commercially available kit (Sigma-Aldrich; Merck KGaA, 387A). RAW 264.7 cells (6x103 cells/well) were plated into 24-well plates and allowed to attach overnight, and 50 ng/ml RANKL was added the next day. RAW 264.7 cells were allowed to differentiate for different numbers of days. After incubation, the RANKL-treated RAW 264.7 cells were fixed with fixative solution (25 ml citrate solution, 65 ml acetone and 8 ml of 37% formaldehyde) at room temperature for 30 sec and TRAP staining solution was added and incubated at 37˚C for 1 h in the dark. The morphology was observed using a Leica DMI6000 B microscope (Leica Microsystems GmbH) at x200 magnification. The number of TRAP-positive multinucleated cells was counted in 20 fields per treatment and the average number of TRAP-positive multinucleated cells in a triplicate experiment are shown in the results.
Western blotting
Cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer with a protease and phosphatase cocktail inhibitor (Thermo Fisher Scientific, Inc.). Cellular debris was cleared from lysates by centrifugation at 14,000 x g for 20 min at 4˚C, and the protein concentration was determined using a BCA protein assay (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of cell lysates containing 30 µg protein were separated on an 12% gel using SDS-PAGE, transferred to a PVDF membrane (Bio-Rad Laboratories, Inc.), blocked with 5% BSA (cat. no. SW3015; Beijing Solarbio Science & Technology Co., Ltd.) for 2 h at room temperature, and then visualized with enhanced chemiluminescence substrate (Pierce; Thermo Fisher Scientific, Inc.) as previously described (14,15). GAPDH was used as a reference protein and lamin was used as a reference for nuclear p65. Quantification of western blotting was performed using ImageJ software (National Institutes of Health).
Cell adhesion assays
RAW 264.7 cells were cultured on a 96-well plate at density of 5x104 cells/well for 3 h. Unbound cells were removed by washing with PBS prior to the addition of 50 µl/well hexosaminidase substrate (3.75 mM 4-Nitrophenyl N-acetyl-β-D-glucosaminide, 0.25% Triton X-100, 0.05 M citrate buffer, pH 5.0). After 2 h, 75 µl/well development buffer (5 mM EDTA, 50 mM glycine, pH 10.4) was added and plates were read at 405 nm as previously described (2,16).
Statistical analysis
Statistical analysis was performed using a two-tailed unpaired Student's t-test or ANOVA with Tukey's post hoc test, and was based on a minimum of three replicates using Prism 5 for Mac statistical software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
RANKL treatment helps to induce cell differentiation (i.e. osteoclastogenesis) (10,17). Fig. 1 demonstrates that the RANKL treatment significantly increased the gene and protein expression of VASP, αV integrin and TRAP in RAW 264.7 cells over 7 days as determined by RT-PCR (Fig. 1A and B) and western blot analysis (Fig. 1B and C), respectively, when compared to basal levels (day 0). However, no significant differences in the expression of the three genes were observed during the 7 days in the negative control group (Fig. S1). Furthermore, the effect of RANKL treatment on osteoclast differentiation and maturation was explored using a TRAP assay. TRAP is an early enzyme marker of osteoclastogenesis, which is readily detectable in mononuclear pre-osteoclasts, through to mature osteoclasts (18). Fig. 1E and F shows the number of TRAP-positive multinucleated osteoclasts differentiated from RAW 264.7 cells in the presence of RANKL for a period of 7 days.
Figure 1
Differentiation of RAW 264.7 cells using RANKL showing a time-dependent increase in the expression of αV-integrin, TRAP and VASP post-RANKL treatment. (A) Quantification and (B) representative image of RT-PCR. (C) Quantification and (D) representative image of western blotting. (E) Representative images following TRAP staining showing the increase in the number of multinucleate cells (x200 magnification). (F) Quantification of the multinucleate cells as determined by TRAP staining. The number of TRAP-positive multinucleated cells was counted in 20 fields/treatment. Data are presented as the mean ± standard deviation (three repeats). *P<0.05 and **P<0.01 compared with day 0. VASP, vasodilator-stimulated phosphoprotein; RANKL, receptor activator of nuclear factor κ-Β ligand; TRAP, tartrate-resistant acid phosphatase.
Knockdown of VASP downregulates the expression of osteoclast-specific genes
VASP has been reported to play an important role in cell behaviour, including in cell differentiation (1,2). To further elucidate the role of VASP in osteoclast differentiation, VASP expression was knocked down in RAW 264.7 cells using si-RNA post-RANKL treatment. Fig. 2 demonstrates a successful VASP-knockdown in the RAW cells as determined at the transcriptional (RT-PCR) and translation (western blotting) level when compared with the mock group.
Figure 2
VASP expression after using si-RNA in RAW 264.7 cells. (A) Quantification and (B) representative image of RT-PCR. (C) Quantification and (D) representative image of western blotting. Data are presented as the mean ± standard deviation (three repeats). ***P<0.001 compared with mock. VASP, vasodilator-stimulated phosphoprotein; NC, negative control; si, small interfering.
Next, the effect of VASP-knockdown on the osteoclast-specific genes in differentiated RAW 264.7 cells was determined. A significant decrease in the gene (Fig. 3A-D) (compared with the mock group) and protein (compared with the siNC group) (Fig. 3E-F) expression of αV-integrin and TRAP was observed in the RANKL-treated VASP-knockdown cells. Furthermore, a significant decrease in multinucleated cell formation was observed in VASP-knockdown cells as determined by the TRAP staining (P<0.05; Fig. 4A and B). A Trypan blue exclusion assay (viability assay) was performed as a control before proceeding to the TARP staining to rule out the possibility of apoptosis or cell death. No significant differences in the viability of cells were observed following VASP-knockdown (Fig. S2). These results demonstrate the role of VASP in the osteoclast differentiation and its effect on osteoclast-specific genes. Future studies should explore the underlying mechanism behind the low level of cell differentiation following VASP silencing.
Figure 3
Effect of VASP silencing. Gene expression of (A) VASP, (B) αV-integrin and (C) TRAP after using siRNA as measured by RT-PCR. (D) Representative gel images of RT-PCR for VASP, αV-integrin, TRAP and GAPDH. Protein expression of (E) VASP, (F) αV-integrin and (G) TRAP after using si-RNA as measured by western blotting. (H) Representative western blotting for VASP, αV-integrin, TRAP and GAPDH. GAPDH was used as the loading control. Data are presented as the mean ± standard deviation (three repeats). ***P<0.001 compared with mock. VASP, vasodilator-stimulated phosphoprotein; TRAP, tartrate-resistant acid phosphatase; NC, negative control; si, small interfering.
Figure 4
Effect of VASP-knockdown on TRAP staining in differentiated osteosarcoma cells. (A) Representative images following TRAP staining showing multinucleate cells (x200 magnification). (B) Quantification of the multinucleate cells as determined by TRAP staining. The number of TRAP-positive multinucleated cells was counted in 20 fields/treatment. Data are presented as the mean ± standard deviation (three repeats). *P<0.05 compared with mock. VASP, vasodilator-stimulated phosphoprotein; TRAP, tartrate-resistant acid phosphatase; NC, negative control; si, small interfering.
VASP-knockdown inhibits lamellipodia protrusion and cell adhesion of osteoclasts
VASP protein acts as an actin anti-capping protein and can therefore regulate the formation of membrane protrusions, such as lamellipodia and filopodia, which consequently affects cell motility and tumour metastasis (19,20). The effect of VASP-knockdown on the morphological changes of lamellipodia protrusion and actin fibbers was determined by microscopy in differentiated murine macrophage RAW 264.7 cells. A decrease in the length of F-actin fibers and lamellipodia protrusion in VASP-knockdown cells compared with their controls was observed (Fig. 5). Additionally, VASP-knockdown significantly reduced the percentage of RAW 264.7 cell adhesion compared with the mock control group (Fig. 6).
Figure 5
VASP-knockdown inhibits lamellipodia protrusion in receptor activator of nuclear factor κ-Β ligand-differentiated RAW 264.7 cells osteoclasts. Fixed cells were stained using fluorescent antibodies as described in the methods section. The fluorescent staining was visualized and imaged using a confocal scanning microscope (x400 magnification; scale bar, 50 µm). VASP, vasodilator-stimulated phosphoprotein; NC, negative control; si, small interfering.
Figure 6
VASP-knockdown inhibits cell adhesion activity of receptor activator of nuclear factor κ-Β ligand-differentiated RAW 264.7 cells. Data are presented as the mean ± standard deviation (three repeats). **P<0.01. VASP, vasodilator-stimulated phosphoprotein; NC, negative control; si, small interfering.
VASP-knockdown reduces the activation of NF-κB signaling
The effects of VASP-knockdown on NF-κB signalling were determined. RANKL, a known activator of the NF-κB pathway triggers a rapid increase and mobilization of NF-κB components, and IκB kinase (IKK) complex formation (7). RANKL treatment increased the phosphorylation of IκBα in a time-dependent manner in RAW 264.7 cells (Fig. 7). Notably, the levels of RANKL-induced IκBα phosphorylation were low in the VASP-knockdown cells compared with the scrambled control cells. Additionally, RANKL-induced nuclear translocation of p65 was significantly reduced in the cells treated with si-VASP. In addition, the expression of NF-κB downstream signalling factors, including c-Fos and NFATc1, was also inhibited in VASP-siRNA cells compared with scrambled control cells. These results confirm the involvement of NF-κB signalling in the modulation of osteoclast differentiation by VASP.
Figure 7
VASP-knockdown inhibits receptor activator of nuclear factor κ-Β ligand-induced activation of NF-κB, cFos and NcFAT1 and phosphorylation of p65 and IκBα in 264.7 cells osteoclasts. Representative images of western blotting for (A) p-IκBα and IκBα, (B) cytoplasmic and nuclear p65, and (C) c-Fos and NFATc1. VASP, vasodilator-stimulated phosphoprotein; TRAP, tartrate-resistant acid phosphatase; NFATc1, nuclear factor of activated T cells cytoplasmic 1; NC, negative control; si, small interfering.
Discussion
Osteoclasts play an essential role in bone homeostasis as characterized by their bone resorptive activity. RANKL, an indispensable cytokine for osteoclastogenesis, activates a series of intracellular signal pathways once it binds to its receptor, which in turn initiates monocyte/macrophage differentiation into multinucleated osteoclasts in vitro (7,21-23). The feasibility to generate osteoclast-type cells in vitro makes it easy for researchers to investigate the molecular mechanisms of this process (7,21-23). In the present study, RANKL-differentiated RAW 276.4 cells significantly increased the expression of VASP, TRAP and αV-integrin. These results are consistent with the previous findings, whereby TRAP mRNA expression has been demonstrated to be upregulated by RANKL treatment in different experimental settings (24). Increased TRAP expression has been used as a marker of bone absorption of bone metastases in cancer (25).VASP plays an important role in cellular activities that depend on cytoskeletal rearrangement. A positive correlation between VASP expression, and pathological stage and metastasis indicates its importance in various pathophysiology (2,5,7,26). Our previous work demonstrated a significant increase in VASP expression in samples from patients with metastatic osteosarcoma compared with patients with non-metastatic osteosarcoma (2). The data from the VASP-knockdown experiment in the present study demonstrated the importance of VASP in the regulation of osteoclast-specific genes in differentiated murine osteoclasts.Osteoclasts are large, multinucleated cells that derive from the monocyte lineage, and express signalling molecules, such as integrins, paxillin, vinculin, talin, protein kinases and actin-associated molecules (27,28). Although mature osteoclasts express numerous integrins, it is considered that integrins of the β1, β2 and αv families are the major adhesion proteins (27,29). Integrins are highly induced in differentiating and mature osteoclasts, and bind to extracellular matrix components. VASP-knockdown significantly reduced the gene and protein expression of the αV-integrin in murine osteoclasts in the current study. These results further indicate the importance of VASP in osteosarcoma metastasis.The importance of VASP proteins in facilitating actin-filament and membrane protrusions is well-established (19). However, the cell motility affected by VASP is not completely established and appears to be cell type-dependent. VASP-knockdown decreased filipodia formation and length of F-actin fibers when compared with the control in the current study. Day 5 was chosen to observe F-actin fibers and lamellipodia protrusion using a confocal microscope, because a significant increase in the expression of osteoclastogenesis genes at day 5 post RANKL treatment has been observed in the present study and other previous studies (11,30,31). These data further support VASP as a positive regulator of osteoclast cell migration. Overexpression of VASP in wild-type NIH 3T3 cells results in metaplasia to cancer cells (32,33). VASP-expressing Mg-63 osteosarcoma cells have high migration capacity compared with reduced VASP-expressing Saos-2 cells (2), which corroborates the results of the present study.RANKL signalling is considered the main target of anti-resorptive agents that suppress osteoclast activation and bone loss. RANKL-induced osteoclast differentiation activates NF-κB and NFATc1. The activation of NF-κB and NFATc1 is mediated by adaptors, including TRAF6 and IKK, that leads to osteoclast differentiation (34). To understand the possible mechanisms by which VASP modulates osteoclast differentiation, the effect of VASP on the NF-κB activation was investigated in RAW 264.7mouse monocyte macrophages. Post-translational modification of NF-κB subfamily proteins is critical in the modulation of NF-κB activation, especially, phosphorylation of p65 and IκB kinase to induce osteoclastogenesis (34,35). Osteoclastogenic genes, such as TRAP, are also activated via this pathway (36). Furthermore, the results of the present study demonstrated that the number of TRAP-positive osteoclasts decreased significantly with silencing of VASP. Additionally, RANKL treatment increased NF-κB expression and phosphorylation of the p65 and Iκk in RAW264.7 macrophages. Pre-treatment with si-VASP significantly inhibited the RANKL-induced expression of NF-κB complex subunits. Consequently, these results demonstrate the importance of NF-κB signaling in the modulation of osteoclast differentiation by VASP in RANKL-stimulated macrophages. In addition to the NF-κB signalling, we have previously demonstrated the role of the c-Fos/NFATc1 pathway in osteoclast development and arrest osteoclastogenesis (16).In the present study, a decrease in the RANKL-induced c-Fos and NFATc1 expression was observed in VASP-knockdown cells. Taken together, the present findings demonstrate that silencing of VASP inhibits RANKL-induced osteoclast differentiation in vitro, and prevents activation of pivotal transcription factors, such as NF-κB, c-Fos and NFATc1. Caution should be extrapolated to interpret the results, as it is unclear whether these changes are directly caused by VASP by regulating the expression of effected proteins or indirectly as a product of upstream or downstream genes in the VASP signalling e.g. cyclic nucleotide-dependent kinases PKA and PKG (2,4,36) of VASP silencing. However, bearing in mind the diversity of the intracellular and intercellular signalling networks that are built around osteoclast maturation and differentiation, targeting VASP more specifically might be promising for the treatment or prevention of osteoclast-related diseases. Therefore, further investigations into the precise mechanisms by which VASP modulates the osteoclastogenesis are required.
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