Animal care
Five-week-old male C57BL/6N mice were purchased from the Orient Bio Inc. (Seongnam, Korea). All mice were maintained under specific pathogen-free conditions and kept at a 12-h light-dark cycle in the animal facility of the Asan Institute for Life Sciences (Seoul, Korea). All mice were provided ad libitum access to rodent chow and water. Euthanasia was performed by an intraperitoneal injection of 40 mg/kg Zoletil 50 (Virbac, Carros, France) and 5.6 mg/kg Rompun (Bayer Korea, Seoul, Korea). No specific criteria were applied for inclusion or exclusion in the animal studies. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Sciences (Approval No. 2018-12-230).
Cell culture and fractionated secretomics
Murine osteoblastic MC3T3-E1 and human leukemic K562 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in a-MEM and RPMI1640 media with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), respectively. K562 cells (3×105/well in 6-well culture plate) were incubated in culture medium, with or without 1 nM PMA (Sigma-Aldrich, St. Louis, MO, USA) for 3 days to collect CMs of pre-MK and MK-like cells, respectively. After changing the media to phenol red-free a-MEM without PMA/FBS, the cells were incubated for 24 h. CMs of pre-MK and MK-like cells were harvested. The CM was centrifuged at 3000 rpm for 10 min (Combi-514R; Hanil Science Industry, Incheon, Korea). To eliminate contaminants, the resulting supernatant was filtered using centrifugal filter units (Millipore, Bedford, MA, USA). The filtered CM was separated into 96 fractions by reverse phase C18 (Vydac 218TP5215, 150 mm × 2.1 mm) chromatography. Then, 500 ml of each fraction was dried and used for cell viability assay. After cell viability assay, the fractions were selected. For LC-MS/MS analysis, 100 mg protein of the fractions were reduced in 10 mM of DTT at 56°C for 20 min and alkylated by incubation in 100 mM iodoacetamide at room temperature in the dark for 15 min. Trypsin digestion was done for 24 h at 37°C and dried. Tryptic digests were analyzed using LTQ Orbitrap Velos (Thermo, San Jose, CA, USA) mass spectrometer equipped with nano HPLC system. The peptide identification was performed using Proteome discoverer Thermo software with human Uniprot database. Instead of primary murine MK cells, K562-derived MK-like cells were used for secretomics to prevent mice’s excessive sacrifice, to exclude contamination by other cells, and to search for a human factor. We validated MK-like cells in this research as previously.13
Purification and differentiation of primary murine MK cells
For MK differentiation, mouse fetal livers of 15.5-day gestational (E15.5) C57BL6 mice were obtained, as described previously.17 Harvested cells were cultured for 3–4 days in DMEM containing 10% FBS and 50 ng/mL murine thrombopoietin (R&D Systems Inc., Minneapolis, MN, USA) and purified using bovine serum albumin (BSA) gradients. After that, MK-enriched (MK-CM) and non-MK (pre-MK CM) fractions were collected and cultured in phenol red-free a-MEM without FBS and thrombopoietin for 24 h. MK-CM and pre-MK-CM were collected via filtration through a 0.45-µm membrane filter and stored at −80 °C until use.
Generation of Vgf -/- mice
Vgf-/- C57BL6 mice were generated and obtained from Genetically Engineered Animal Research (GEAR) Core, Asan Medical Center (Seoul, Korea). As described previously, using TALEN methodology, Vgf-TALEN mRNA was injected into C57BL6 mouse embryos.18 For genotyping, genomic DNAs were extracted from tail tissues and used in PCR with a Solg Taq DNA Polymerase kit (SolGent, Daejeon, Korea), using the forward (5’-TCC TTC TAC TGA TCC AGG GGT-3’) and reverse (5’-GTC ATC CTT TGG CCG GGA C-3’) primers.
Cell viability and proliferation assays
Murine pre-osteoblast MC3T3-E1 cells (5×103 cells/well) were seeded into 96-well culture plates and incubated for 1–2 days. For the cell viability assay, 10 μL Cell Counting Kit-8 solution (Dojindo, Kumamoto, Japan) was added to each well, and the plates were incubated for 1 h. A microplate reader (Tecan Group Inc., Mannedorf, Switzerland) was used to measure absorbance at 450 nm. For the cell proliferation assay, the cells were incubated with 5-bromo-2′-deoxyuridine (BrdU) for 24 h, and cell proliferation was analysed using a BrdU labelling and detection kit (Roche, Mannheim, Germany) according to the manufacturer's instructions.
Bone nodule formation assay
Using 14-day treatment with 50 μg/mL ascorbic acid (Sigma-Aldrich) and 10 mM β-glycerophosphate (Sigma-Aldrich), MC3T3-E1 cells let to differentiate into osteoblasts. The cells were fixed with 70% ethanol, stained with 40 mM alizarin red S solution (Sigma-Aldrich), and washed with distilled water. After dissolving the bound alizarin red S solution was dissolved with 10% cetylpyridinium chloride (Sigma-Aldrich), we quantified the eluted samples by measuring absorbance at 570 nm using a microplate reader (Tecan Group Inc).
VGF-derived peptides and antibodies
The VGF-derived peptides used in this study include NERP1 (#076-89), NERP2 (#076-91), AQEE19 (#007-73), AQEE30 (#007-70), TLQP21 (#003-90), and TLQP62 (#007-82) from Phoenix Pharmaceuticals (Burlingame, CA, USA). Mouse AQEE30 and H2N-8-amino-3,6-dioxaoctanoic acid (mini-PEG2)-conjugated AQEE30 (PEG2-AQEE30) were synthesized from Peptron Inc. (Daejeon, Korea). We used the following antibodies; VGF (ab69989) antibody from Abcam (Cambridge, UK); p21 (sc-6246) antibody from Santacruz Biotechnology (Dallas, TX, USA); His (12698), pRb (9308), and Rb (9309) antibodies from Cell signaling (MA, USA); a-tubulin (T9026) and actin (A3854) antibodies from Sigma-Aldrich; osteocalcin (M173) antibody from Takara (Tokyo, Japan); and C3AR1 (orb101135) antibody from Biorbyt (Cambridge, UK).
Osteocalcin and AQEE30 ELISA assays
MC3T3-E1 cells (2×105 cells/well) were seeded in 12-well culture plates and differentiated into osteoblasts with 5 mM AQEE30 for 7 days. CM was harvested and measured using an osteocalcin ELISA kit (BT-470, Alfa Aesar, Ward Hill, MA, USA). AQEE30 levels in the pre-MK-CM and MK-CM were measured using an AQEE30 ELISA kit (Phoenix Pharmaceuticals) according to the manufacturer's instructions.
Cell-cycle assay
MC3T3-E1 cells (1×105 cells) were seeded into 35-mm culture dishes, cultured for 1 day, and treated with 1 mM AQEE30 for 12 h before trypsinization, washing with PBS, and fixation with 70% ethanol for 1 h. The cells were centrifuged at 1700 rpm for 5 min, washed with PBS, and treated with 50 mg/mL RNase solution (Sigma-Aldrich) at room temperature for 15 min. The samples were treated with 50 mg/mL propidium iodide solution (Sigma-Aldrich) at room temperature for 30 min and measured by a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA)
Micro-computed tomography (Micro-CT)
Femurs of Vgf-/- and their wild-type (WT) littermate mice were fixed with 4% paraformaldehyde. Bone samples were scanned with Skyscan 1172 system (Skyscan, Belgium) at 70 kV, 126 mA, and 6.8-mm pixel size. The regions of interest extended 3 mm from the growth plate of each femur to the proximal metaphysis, and three-dimensional algorithms were used to determine the relevant morphometric parameters using CT-analyser (version 1.16.1.0; CTAn, Skyscan) software.
Immunofluorescence, dynamic histomorphometry, and serum bone marker
For immunofluorescent staining, the dissected femur or calvaria bone tissues were fixed, decalcified, and sectioned with a cryostat; the 5-μm-thick paraffin bone sections were deparaffinized, rehydrated, and stained with the anti-osteocalcin antibody (1/200 dilution; Takara) for 16 h at 4°C. Then, the sections were washed and incubated with an Alexa Fluor 647 secondary antibody (1/400 dilution; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Nuclei were counterstained with DAPI, and fluorescence was detected using a confocal microscope (LSM710; Carl Zeiss, AG, Oberkochen, Germany). For dynamic histomorphometry, Vgf-/- mice and their WT littermates were injected with calcein (30 mg/kg; Sigma-Aldrich) 5 and 3 days before they were sacrificed. The femurs were cut into 20-mm-thick sections, and the bone sections were photographed and analysed with a semiautomatic image-analysing system (Histometry RT Digitizer; System Supply Ltd.) and the Bio-Quant program (version 19.9.60; Bio-Quant, Inc., Nashville, TN, USA). For the serum bone marker analysis, serum osteocalcin was measured using an osteocalcin ELISA kit (BT-470, Alfa Aesar, Lancashire, UK).
In vivo calvaria bone formation
Calvaria bone formation assay was performed according to the previous report with minor modifications.13 Five-week-old male C57BL/6N mice were injected subcutaneously over the right parietal calvarial bone with AQEE30 and PEG2-AQEE30 at a dose of 0.5 mg/kg in 50 μl PBS daily for 5 days. After 16 days, all mice were euthanized and calvaria were harvested. Calvaria were fixed with 4% paraformaldehyde for 24 h and decalcified in Osteosoft solution (Sigma-Aldrich, 1.01728.1000) for 2 weeks. Paraffin-embedded sections (5 μm-thickness) of calvaria were stained for hematoxylin and eosin (H&E) or were processed for immunostaining of osteoclacin as described above. Images were acquired with an Olympus VS200 slide scanner (Olympus, Tokyo, Japan) and were analyzed with an Olympus OlyVIA software (version 3.3; Olympus). Calvaria bone width was evaluated as the length including both old and new bones. The calvaria bone width was determined by averaging four lengths measured at equal intervals from 900 to 1,700 mm from the sagittal suture. Total bone area was determined in the area between the sagittal suture and the muscle-attached site. New bone area was measured on the periosteal surface of the calvaria in the same region for total bone area. Width and bone area measurements were analyzed using Image Pro Plus program (version 6.0; Media Cybernetics, Bethesda, MD, USA).
RNA interference, real-time PCR, and western blot
VGF and C3AR1 siRNAs (listed in Supplementary Table 1) were purchased from Qiagen (Valencia, CA, USA) and Bioneer (Daejeon, Korea), respectively. According to the manufacturer's instructions, MC3T3-E1 cells were transfected with 20 pmol siRNAs for 24 h using Lipofectamine 2000 (Invitrogen). Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green I Master Mix (Roche) according to the manufacturer’s instructions, and primers used in qRT-PCR are listed in Supplementary Table 2. TaqMan probes (Invitrogen) of Mm00443947_m1, Mm00726334_s1, and Mm03928990_g1 were used to determine the expression of CDK2, CDK4, and 18S, respectively.
For western blot, cells were lysed with a RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, 1% SDS, 1 mM PMSF, 1 mM Na3VO4, and 1 mM glycerol phosphate) that contained a protease inhibitor cocktail (Sigma-Aldrich). The cell lysates were centrifuged at 13,000 rpm for 10 min, and the supernatants were denatured with an SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2.5% SDS, 10% glycerol, 0.002% bromophenol blue, and 5% mercaptoethanol) and subjected to SDS-PAGE. The separated proteins were transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA) to be probed with primary and HRP-conjugated secondary antibodies. All blots were developed with an ECL kit (Perkin Elmer, Waltham, MA, USA), and the intensity of the western blotting bands was quantified by Image J (version 1.52a; NIH, Bethesda, MD, USA).
AQEE30 and C3AR1-binding ELISA
The binding ELISA was performed as described previously.19 MC3T3-E1 cell lysates were added to 10 μg/mL AQEE30 or BSA-coated binding ELISA 96-well plates (Thermo Scientific) for 2 h treatment at room temperature. The plates were washed, blocked with 1% BSA, and then incubated with the C3AR1 antibody (Biorbyt) for 2 h. After washing, an HRP-conjugated secondary antibody (Cell Signaling Technology) was added for 2 h. The HRP-TMB (3,3′,5,5′-tetramethylbenzidine) system generated the colorimetric signal. The absorbance was read at 450 nm using an ELISA microplate reader (Tecan Group Inc.).
Peptide pull-down assay
The peptide pull-down assay was performed as described previously.20 His-tagged C3AR1 (Sino Biological, HG11469-CH, Beijing, China) were expressed in HEK293T cells (American Type Culture Collection), and cell lysates were prepared with a RIPA buffer, mixed with 10 mg and 30 mg biotinylated AQEE30 peptide that had been previously bound to streptavidin agarose beads (Thermo Scientific) at 4°C in binding buffer (20 mM Tris-HCl, pH 7.3, 150 mM KCl, 0.2 mM EDTA, 20% glycerol, and 0.1% Nonidet P-40), and incubated overnight. The beads were washed, and bound proteins were analysed by western blot using the anti-His antibody.
cAMP ELISA and protein kinase A activity assay
MC3T3-E1 cells were pre-treated with 1 μM SQ22536 (Sigma-Aldrich, S153), 1 μM H89 (Sigma-Aldrich, B1427), or 1 μM SB290157 (Sigma-Aldrich, SML1192) for 1 h, and 5 μM AQEE30 was added for 30 min. Cell lysates were lysed with 100 μL lysis buffer (Enzo Laboratories, Farmingdale, NY, USA). After centrifugation at 13,000 rpm for 10 min, the supernatant was analysed with a cAMP ELISA kit (Enzo Life Sciences) according to the manufacturer's instructions. The absorbance was measured at 405 nm using a microplate reader (Tecan Group Inc). For the PKA activity assay, total cell lysates were prepared and examined by a PKA activity assay kit (Enzo Life Sciences) according to the manufacturer's instructions, and absorbance at 450 nm was measured using a microplate reader (Tecan Group Inc.).
Quantification and statistical analysis
All quantitative data represent mean ± SEM. Intergroup differences were examined using a Student’s two-tailed t-test, and multigroup differences were ascertained using a one-way analysis of variance followed by the Bonferroni multiple-comparisons test. All statistical analyses were performed using GraphPad Prism (version 8.0; GraphPad Software Inc., San Diego, CA, USA), and p < 0.05 was considered statistically significant.