Cell culture and osteoclast differentiation
The mouse macrophage cell line RAW264.7 (ATCC, Cat# TIB-71™) and human kidney epithelial cell line 293T (ATCC, Cat# CRL-3216™) were cultured in DMEM (HyClone, Cat# SH30243.01B) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 atmosphere. All cell lines were routinely screened for mycoplasma contamination using a Mycoplasma Stain Assay Kit (Beyotime, Cat# C0296).
Bone marrow macrophages (BMMs) were isolated from 4-week-old male C57BL/6J mice as previously described [44, 45]. In short, the femur was separated under aseptic conditions, and the bone marrow cavity was flushed with α-MEM (HyClone, Cat# SH30265.01B) containing 10% FBS and 1% penicillin–streptomycin. The cell suspension was cultured in complete α-MEM at 37 °C in a 5% CO2 atmosphere overnight to filter off adherent mesenchymal stem cells. The supernatant was transferred to a new cell culture flask, M-CSF was added to a final concentration of 50 ng/ml, and the cells were cultured for another 3 days to allow BMMs to adhere and proliferate.
For OC differentiation, cells were cultured in complete α-MEM containing 30 ng/ml M-CSF (R&D Systems, Cat# 416-ML-010) and 50 ng/ml RANKL (R&D Systems, Cat# 462-TEC-010) for 6 days. The medium was changed every other day. Multinucleated osteoclasts were fixed in 4% paraformaldehyde at room temperature for 20 min, soaked in PBS containing 0.5% Triton-X for 30 min, and stained with tartrate-resistant acid phosphatase (TRAP) solution (Servicebio, Cat# G1050) at 37 °C for 50 min. Osteoclast differentiation was also confirmed by immunofluorescence staining with DAPI (Servicebio, Cat# G1012) and phalloidin (Servicebio, Cat# G1041). Fluorescent images were acquired using an inverted microscope system (Olympus, IX73, Japan). Cells with 3 or more nuclei were counted. Statistics are based on the number of OCs in 8 fields. For drug experiments, 10 µM NFAT Inhibitor (MCE, Cat# HY-P1026) was used to inhibit NFATc1, and 10 µM T-5224 (MCE, Cat# HY-12270) was used to inhibit c-Fos.
Isolation, culture and differentiation of bone mesenchymal stem cells (BMSCs)
Primary BMSCs were collected from 4-week-old Wt and Bhlhe40-/- male mice as previously described [46]. In brief, the mechanically separated femoral and tibial diaphysis were digested with α-MEM containing 1 mg/ml collagenase II (Gibco, Cat# 17101015) and 2 mg/ml 100 mg dispase II (Millipore Sigma, Cat# D4693) for 2 h. Cells were cultured in complete α-MEM at 37 °C in a 5% CO2 atmosphere. The 3rd to 5th generation BMSCs were used in this study.
For osteoblast differentiation, cells were cultured in α-MEM containing 10% FBS, 10 nM dexamethasone (Sigma-Aldrich, Cat # D2915), 10 mM β-glycerophosphate (Sigma-Aldrich, Cat #G6251) and 50 µM L- Ascorbic acid (Sigma-Aldrich, Cat# A4403). ALP (Beyotime, Cat# C3206), Alizarin Red S(ARS) (Sigma-Aldrich, Cat# A5533; 1% solution in water; pH 4.2) and Von Kossa staining (Servicebio, Cat# GP1054) were performed at 7 days, 21 days and 28 days, respectively. Microscopy images were obtained using a 10 × phase-contrast objective on an Olympus IX73 microscope.
The proliferation and apoptosis assay
MTT was used to detect the proliferation of BMMs after knockdown or overexpression of BHLHE40. Inoculate treated BMMs (1 × 105 cells/ml) in a 96-well plate. Theer days later, 100ul of complete α-MEM containing 10% MTT buffer (Procell, Cat# PB180519) was added to each well and incubated at 37 °C for 4 h. After removing the supernatant, 100 µl of dimethyl sulfoxide (DMSO) was added to the wells, and a multifunctional microplate reader (PE Enspire) was used to detect the absorbance at a wavelength of 492 nm.
For the apoptosis assay, BMMs with knockdown or overexpressed of BHLHE40 and their controls were gently dissociated with trypsin without EDTA, and apoptosis was detected with an Annexin V-FITC/PI apoptosis kit (MultiSciences, Cat# 70-AP101-100) according to the manufacturer's instructions. CytoFlex S flow cytometer and Cytexpert analysis software (Beckman Coulter) were used for analysis.
Mice and bone marrow transplantation
Bhlhe40−/−(BHLHE40 KO) mice were a gift from the Collaborative Innovation Center of Model Animal Wuhan University. Targeting of the Bhlhe40 locus was performed by standard techniques. The BssHII–BssHII genomic fragment of Bhlhe40 as previously described [47], which contains the promoter and coding region including the ATG in exon 1, was replaced with a Neo cassette. The resulting chimeric mice were backcrossed to C57BL/6 mice more than six to eight times.
For elderly animal models, 1-month-old or 12-month-old male mice (C57BL/6) were sacrificed, and the femurs were isolated and fixed with 4% paraformaldehyde for subsequent experiments.
To establish an osteoporosis mouse model [48], 6-week-old female mice (C57BL/6) were anesthetized with 1–4% isoflurane. A 0.5 cm single midline dorsal incision was made in the lower back through the skin. The connective tissue under the skin was gently freed, the ovary was positioned under the thin muscle layer, and a small incision was made on each side to enter the abdominal cavity of the skin. Expose fallopian tubes and ovaries. The ovaries were identified and placed back into the abdominal cavity (Sham). Ligation was performed around the fallopian tube and small sterile scissors were used to gently cut off the fallopian tube to remove the ovaries. The rest of the fallopian tube was placed back into the abdominal cavity (OVX) and sutured layer by layer.
Since adult osteoclasts are mainly derived from HSCs [6], this allows us to specifically study the effect of gene knockout in osteoclasts on bone mass. Bone marrow transplantation was performed as previously described [49] and grouped as shown in Fig. 4A. In brief, 6-week-old male recipient mice (Wt and Bhlhe40−/−) were lethally irradiated (8 Gy). After 4 h, the donor mice (4-week-old Wt and Bhlhe40−/−) were sacrificed and the femurs were separated. The bone marrow was collected from the femur by washing 3 times with PBS supplemented with 1% FBS and filtering the cells through a 200 mesh screen. The cells were washed in PBS and resuspended at a concentration of 107 cells/ml. Then, 106 donor cells (100 μl) were injected into the tail vein of each mouse in the recipient group, and the animals were kept for 7 weeks. Peripheral blood was collected (50 µl) to extract DNA for chimerism identification by PCR. The primer sequences used in the assay are listed in Additional file 8: Table S1. No statistical method was used to predetermine the sample size of animal studies. No mice were excluded from analyses except those with unsuccessful transplantation.
Knockdown and overexpression
To knock down Bhlhe40, Nfatc1 and Fos, a riboFECT CP Transfection Kit (RiboBio, Cat # R10035.7) was used for siRNA transfection for 24 h. The gene sequences used for the target siRNA are listed in Additional file 8: Table S1. The Bhlhe40 overexpression plasmid was purchased from Shanghai Genechem. Lentivirus packaging and infection were performed as previously described [50]. The plasmids pSPAX2, pMD2.G and overexpression plasmids were cotransfected into 293 T cells to package lentivirus. The supernatant was collected at 48 and 72 h and lentiviruses were concentrated (Millipore, Ultracel-100 regenerated cellulose membrane, 15 mL sample volume, Cat# UFC9100). Lentiviral infection (MOI = 100) of BMMs was performed in the presence of 10 µg/ml polybrene for 24 h. Cells were selected with 2 µg/ml puromycin for 72 h. The knockdown efficiency or overexpression level of Bhlhe40 was verified by qPCR. The knockdown efficiency levels of Nfatc1 and Fos were verified by qPCR.
Chromatin immunoprecipitation (ChIP)
The samples were prepared as described previously [51]. BMMs were cultured in 30 ng/ml M-CSF for 3 days and fixed in 1% formaldehyde for 10 min at room temperature. Cells were neutralized with glycine and collected to make cell lysates. Cells were resuspended in sonication buffer at 4 °C for 10 min, and then each sample was sonicated 6 times with 40% AMP for 15 s. After centrifugation at 8000 g for 30 s at 4 °C, chromatin was collected from the supernatant, and 50 μl aliquots of the sample were used to extract the input DNA. After incubating with 1 μl of 1 mg/ml RNase A (Sigma-Aldrich, Cat# R6513) and 2 μl of proteinase K (20 mg/ml) at 65 °C overnight, the input DNA was purified using a TIANGEN Common DNA Product Purification Kit (TIANGEN, Cat# DP204). The chromatin samples were incubated with 1 mg/ml BSA and 40 μl prebalanced protein A/G magnetic beads (MCE, Cat# HY-K0202) for 2 h at 4 °C. After the beads were removed, 1 μg of anti-BHLHE40 antibody was added, incubated overnight and then precipitated with protein A/G magnetic beads. The beads were washed, and DNA was eluted and purified with a TIANGEN Common DNA Product Purification Kit after RNase A treatment. The immunoprecipitated DNA fragments were collected for qPCR amplification. Normal rabbit IgG was used as a negative control. DNA samples were analysed by real-time PCR, and IgG was used for standardization. The nucleotide sequence of the oligonucleotide used in the assay is shown in Additional file 8: Table S1. The antibodies used were summarized in Additional file 9: Table S2.
RNA extraction and quantitative PCR
TRIzol (Thermo Fisher, Cat# 15596026) was used to extract total RNA from BMMs. The reverse transcriptase kit (Vazyme, Cat# R223) was used to reverse transcribe an aliquot of 200 ng total RNA into cDNA. SYBR green mixture (Vazyme, Cat# Q311) and Monad Real-Time PCR instrument (Monad q225) were used for quantitative PCR. The primers used for specific transcripts are listed in Additional file 8: Table S1. The mRNA level was normalized to that of GAPDH.
Micro-CT analysis
All mice were euthanized with CO2, and the femurs were separated and evaluated by the SkyScan 1176 high-resolution micro-CT imaging system (Bruker, Germany). According to the manufacturer's instructions [49], each femur was scanned separately at 55 kV and 200 μA using a 0.25-mm aluminium filter to obtain an isometric resolution of 7-μm. NRecon (Bruker, Germany) was used to reconstruct the image and CTAn (Bruker, Germany) was used for quantitative analysis. The volume of interest (VOI) contains most of the metaphysis and part of the diaphysis. CTVol software was used (Bruker Micro-CT, Germany) to adjust the 3D model. Trabecular or cortical bone parameters in an area from 0.2 mm to 2.3 mm or 4.0 mm to 5.4 mm below the growth plate of the femur were measured.
Mechanical testing
The soft tissue was removed from the femur to ensure consistency between the conditions used in the mechanical test. All 8 samples were mechanically tested in the vertical direction. Polymethyl methacrylate rigid base (PMMA, Aladdin, Cat# 9011-14-7) was used to fix both sides of the femur and contact the metal base [52]. The dynamic fatigue testing machine (BOSE ElectroForce 3220 225N) was brought in contact with the PMMA bases on both sides until nominal compression (< 10 N) was observed. Then, the mouse femur was compressed at a speed of 1 mm/s until the applied load dropped significantly (loss of 50% or more). The mechanical test was carried out at room temperature. All test data were collected at 20 Hz.
Skeletal preparation and staining
As mentioned earlier [53], mice were sacrificed by CO2 and skin and muscles were removed. After fixation in 95% ethanol overnight, remaining samples were transferred to acetone for 48 h. Cartilages were then stained in 0.3% Alcian Blue (Sigma-Aldrich, Cat# A5268) overnight at room temperature. The samples were incubated overnight in 95% ethanol to decolorize and incubated with 1% KOH solution at room temperature for 1 h to remove residual tissue. The KOH solution was removed and incubated with 0.1% ARS solution (Sigma-Aldrich, Cat# A5533) overnight at 4 °C for bone staining. Specimens were incubated in 1% KOH until the excess ARS and tissues were completely cleared, and then stored in glycerol for pictures by Nikon camera.
Immunohistochemistry and histological analysis
For paraffin sections, femurs were fixed in 4% paraformaldehyde for 48 h, decalcified in 10% EDTA for 28 days, and then embedded in paraffin after procedural dehydration. The tissue was sliced (8 μm) with a Leica RM2235 microtome, deparaffinized, and stained with TRAP (Servicebio, Cat# G1050), Masson (Servicebio, Cat# GP1032), Goldner (Servicebio, Cat# GP1053) and haematoxylin–eosin (H&E).
For frozen sections, femurs were fixed in 4% paraformaldehyde for 6 h and decalcified as previously described [54]. After the samples were washed, they were soaked in PBS containing 30% sucrose at 4 °C for 1–2 days, and embedded in a gel-filled disposable histological plastic mold. We used a Leica CM3050S cryostat to slice the samples (20 μm) at − 25 °C.
For immunohistochemistry, sections were deparaffinized and treated with citrate buffer solution (pH 6.0) 3 times at 95 °C, and treated with 3% H2O2 for 20 min at room temperature. Samples were then blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h, and incubated with the primary antibody overnight at 4 °C. After the samples were washed, the Polink-2 Plus polymer HRP detection system (ZSGB-BIO, Cat# PV6001) was used for the secondary antibody incubation of the samples, and DAB (ZSGB-BIO, Cat# ZLI-9017) was used for color development. Hematoxylin was used for nuclear staining. After dehydration and fixation, the slices were scanned by the Aperio VERSA 8 (Leica, Germany) scanner.
Immunofluorescence staining
BMMs and RAW264.7 cells cultured in complete α-MEM containing 30 ng/ml M-CSF and 50 ng/ml RANKL for 0, 2, 4, or 6 days were fixed with 4% paraformaldehyde for 20 min and permeabilized with PBS containing 0.5% Triton-X for 30 min. For anti-BHLHE40 immunofluorescence staining, cells were blocked with PBS containing 5% BSA after fixation and permeabilization, and cells were incubated with BHLHE40 primary antibodies overnight at 4 °C. Then, the cells were incubated with secondary antibodies for 1 h. The images were taken by confocal microscopy (Leica SP8, Germany) and analysed with Leica Application Suite X (LAS X).
For immunofluorescence, before staining with antibodies, the sections were allowed to equilibrate for 30 min at room temperature and rehydrated with PBS at room temperature 3 times for 5 min. After the samples were permeabilized or blocked with PBS containing 0.2% Triton-X 100 or 5% BSA for 1 h, the sections were incubated with the primary antibody overnight at 4 °C. After washing with PBS 3 times for 5 min, sections were incubated with fluorescently labeled secondary antibodies for 1 h. Images were captured with a confocal microscope (Leica SP8) and analysed with LAS X. Sections were also stained with integrin alpha-M (CD11b) (BMMs marker), CTSK (OCs marker) and DAPI (nuclear).
The primary and secondary antibodies used are listed in Additional file 9: Table S2.
Transcriptomic analysis
BMMs were isolated from Wt and Bhlhe40−/− mice, cultured with 50 ng/ml M-CSF for 3 days, and harvested in a GenCatch TM Total RNA Extraction Kit (Epoch Life Sciences, Cat# 1660050) 48 h after OC differentiation. Two micrograms of RNA was used for cDNA library preparation and sequencing following the manufacturer's instructions (HiSeq 2500, Illumina). HISAT2 [55] was used to map sequencing reads to the mouse genome and SAMtools [56] was used to aggregate tag counts at the gene level allowing only one read per position per length. DESeq2 [57] was used to determine the differentially expressed genes. ClusterProfiler [58] was used to perform gene set enrichment analysis, including GO and KEGG analyses. GOplot [59] was used for the visualization of GO analysis. The RNA-seq data in this study are available in SRA, and the BioProject accession number is PRJNA763091 (http://www.ncbi.nlm.nih.gov/bioproject/763091).
Bioinformatics analysis
To explore the potential correlation between Bhlhe40 and OC differentiation, GSE54779 (GPL6246 Affymetrix mouse gene 1.0 ST array) dataset from the Gene Expression Omnibus (GEO) repository at the National Centre of Biotechnology Information website (https://www.ncbi.nlm.nih.gov/geo/) was analysed. This dataset contains three BMM samples treated with RANKL and M-CSF and three BMM samples treated with M-CSF alone. All raw expression data were standardized by R and the Bioconductor (http://www.bioconductor.org) Affymetrix package. The Linear Array Microarray Analysis (Limma) software package was used to identify the differentially expressed genes (DEGs) in BMM samples treated with M-CSF and RANKL compared with samples stimulated with M-CSF alone. Values of p < 0.05 and |log2FC|> 0.5 were considered thresholds for identifying DEGs.
In order to determine the correlation of Bhlhe40 and Fos or Nfatc1, GSE7158 or GSE51495 from the GEO repository at the National Center for Biotechnology Information website (https://www.ncbi.nlm.nih.gov/geo/) was used for analysis (GPL570 Affymetrix Human Genome U133 Plus 2.0 Array or GPL6104 Illumina humanRef-8 v2.0). GraphPad Prism 7.0 was used to analyse the correlation between Bhlhe40 and Fos or Nfatc1 expression in each sample.
The BHLHE40 target genes were predicted through the Gene Transcription Regulation Database (GTRD (http://gtrd.biouml.org/)). The binding sites of BHLHE40 were predicted through the JASPAR database (http://jaspar.genereg.net/).
Statistical analysis
Data were expressed as the mean ± SD. The results were considered statistically significant if the P value was less than 0.05. The random number method was used for random assignment. Researchers blinded to group assignment during the experiment. Individual data points were shown, and the number of samples or images analysed was indicated in the figure and/or legend. The data were analysed using the appropriate Student's t test when comparing two groups or one-way analysis of variance when comparing more than 2 groups. The data points correspond to independent experiments. The variances between the compared groups were similar. All statistical tests were performed using Prism 7.0 software.
