CCL3 in the bone marrow microenvironment causes bone loss and bone marrow adiposity in aged mice

The central physiological role of the bone marrow renders bone marrow stromal cells (BMSCs) particularly sensitive to aging. With bone aging, BMSCs acquire a differentiation potential bias in favor of adipogenesis over osteogenesis, and the underlying molecular mechanisms remain unclear. Herein, we investigated the factors underlying age-related changes in the bone marrow and their roles in BMSCs’ differentiation. Antibody array revealed that CC chemokine ligand 3 (CCL3) accumulation occurred in the serum of naturally aged mice along with bone aging phenotypes, including bone loss, bone marrow adiposity, and imbalanced BMSC differentiation. In vivo Ccl3 deletion could rescue these phenotypes in aged mice. CCL3 improved the adipogenic differentiation potential of BMSCs, with a positive feedback loop between CCL3 and C/EBPα. CCL3 activated C/EBPα expression via STAT3, while C/EBPα activated CCL3 expression through direct promoter binding, facilitated by DNA hypomethylation. Moreover, CCL3 inhibited BMSCs’ osteogenic differentiation potential by blocking β-catenin activity mediated by ERK-activated Dickkopf-related protein 1 upregulation. Blocking CCL3 in vivo via neutralizing antibodies ameliorated trabecular bone loss and bone marrow adiposity in aged mice. This study provides insights regarding age-related bone loss and bone marrow adiposity pathogenesis and lays a foundation for the identification of new targets for senile osteoporosis treatment.

sections of each staining were analyzed per animal. The sampling site for the distal femoral metaphysis was located 0.25-1.25mm proximal to the growth plate and 0.1mm from cortical bone. Images were analyzed using ImageJ in a blinded manner.
For AR staining and quantification, cells were fixed with 4% paraformaldehyde at 4°C and then incubated in 40 mM AR solution (pH 4.2) for 30 min at 37°C.
After imaging, decalcification was performed in 0.1 M HCl for 8 h at 4 °C. Then 10 μl of samples were mixed with 0.5 mL of methyl thymol blue and 0.5 mL of alkaline solution. Absorbance was quantified at 610 nm.
For Oil red O staining, cells were fixed with 4 % paraformaldehyde and stained in Oil Red O solution for 1 h in a 60 °C water bath.

qRT-PCR
Total RNA was prepared using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Single-stranded cDNA was reverse transcribed from 1 μg total RNA using oligo-dT primers. Quantitative PCR was performed on an ABI Prism 7500 system (Applied BioSystems, Foster City, CA, USA) employing SYBR Green PCR Master Mix (Takara Bio Inc., Otsu, Japan). Cycling conditions was as follows 94 °C, 5 s; 60 °C, 34 s; and 72 °C, 40 s for 40 cycles. Actb (β-actin) and Hprt were used as internal controls. Primer sequences were listed in Table   S1.

Western blot
Total protein was prepared by lysing cells on ice for 30 min in a buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors). The proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and detected using anti-CCL3 (AF450, R&D systems),

Luciferase reporter assay
Cells were cultured in 24-well plates. All plasmids were prepared using QIAGEN plasmid purification kit. Transient transfection was performed using Lipofectamin 3000 (Invitrogen) and phRL-SV40 vector (Promega, Madison, WI, USA) was used as transfection efficiency control. Forty-eight hours after transfection, the cells were lysed and both firefly and renilla luciferase activities were evaluated using Dual-luciferase reporter assay system (Promega).

In vitro methylation assay
M.SssI (New England Biolabs, Ipswich, MA, USA) (2 U/μg DNA) was employed to methylate CpG sites in vitro for 6 h at 37 °C and then inactivated for 15 min at 65 °C. Methylated DNA fragments and vectors were ligated and purified by phenol/chloroform extraction and ethanol precipitation.

Osteoclast differentiation assay
Animals were euthanized using isoflurane inhalation anesthesia followed by cervical dislocation. The femur and tibiae of mice were washed with serum-free α-MEM. Bone marrow cells were incubated with α-MEM supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml) for 24 h. Non-adherent cells were harvested, and induced to bone marrow monocytes (BMMs) using M-CSF (20 ng/ml). Cells were cultured in the humidified atmosphere at 37 °C and 5% CO2.
BMMs were incubated in 48-well plate in α-MEM containing M-CSF (20 ng/ml) plus RANKL (100 ng/ml) to form mature osteoclasts. Osteoclasts differentiation was observed by Tartrate resistant acid phosphatase (TRAP) staining. TRAP+ cells with more than three nuclei were considered as mature osteoclasts. TRAP+ cells with more than five nuclei were considered as large osteoclasts.

Immunohistochemistry
Femurs were fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin. Serial sections were cut at 5 μm thickness. Slides were incubated with primary antibody against CCL3 (AF450, R&D systems), DKK-1 (MAB1765, R&D systems) and active β-catenin (19807, Cell Signaling Technology) overnight at 4 °C. For immunohistochemical staining, a horseradish peroxidase-streptavidin detection system (Dako) was used to detect immuno-activity. Isotypoe IgG was used as s negative control (Fig. S19). Five slides in each sample were selected to enumerate the stained cells per ×100 optical fields under a light microscope.       Data were shown as the means ± s.d. ns: no significance.

C/EBPα promoter sequence
Transcription start site Putative STAT3 binding site ChIP region The DNA sequence of -1249bp~+17bp around C/EBPα transcription start site was shown. Transcription start site was labeled in red, putative STAT3 binding site was labeled in blue and ChIP region was labeled in green.           100 μm. All the data were obtained from three independent experiments. Data were shown as the means ± s.d. *: p<0.05, **: p<0.01, ns: no significance.

Figure S17
Figure S17: Histological evaluation of core organs of aged mice in response to CCL3 neutralization antibody administration. Core organs including heart, liver, spleen, lung, and kidney were recovered from aged mice in response to CCL3 neutralization antibody or vehicle administration. H&E staining was employed to evaluate the histological phenotype of these organs described above. All the data were obtained from three independent experiments. Data were shown as the means ± s.d. *: p<0.05, **: p<0.01, ***: p<0.001.