ROSA26-Wnt7b^flox/flox mice were provided by Prof. Fanxin Long from University of Pennsylvania. LysM-Cre or RANK-Cre were purchased from Biocytogen Co. Ltd., Beijing, China. All animal procedures were approved by the Ethical Committees of the State Key Laboratory of Oral Diseases, Sichuan University. The research was carried out in accordance with accredited guidelines. Genotype was identified by PCR analysis with primers for ROSA26-Wnt7b^flox/flox, LysM-Cre, and RANK-Cre.

All procedures were performed according to reported methods. The right femurs were isolated, fixed in 4% paraformaldehyde, and maintained in phosphate-buffered saline (PBS) containing 0.4% paraformaldehyde. All radiographic data were acquired via VIVA 40CT 64GB (Scanco Medical AG, Bassersdorf, Switzerland) system using previously established parameters as follows: X-ray tube potential, 55 kVp; X-ray intensity, 145 μA; integration time, 200 ms; and threshold, 220 mg/cm³. The percentage of bone volume (BV/TV), trabecular thickness (tb.Th.), trabecular number (tb.N.), trabecular separation (tb.Sp), and cortical thickness (ct,Th.) were conducted, and 3D images were reconstructed for visualization.

Eight-week-old mice were labeled with calcein (10 mg/kg, sigma) and alizarin red (30 mg/kg, Sigma) through intraperitoneal injection at 5 and 3 days before sacrificed. Undecalcified femurs were fixed with 4% paraformaldehyde and dehydrated with 30% sucrose dissolved in PBS. Twenty-micrometer sections were performed for microscopy analysis. Images were captured under 510–550 nm and 450–480 nm fluorescent light to image calcein and alizarin. Image processing was used Image Pro Plus software.

Mice 6 and 8 weeks old were fasted for at least 6 h; blood was drawn, allowed to clot at room temperature for 30 min, and then centrifuged at 3,000 rpm for 10 min at 4°C. Serum samples were stored at −80°C. ELISA was employed to determine mouse serum PINP and CTX-I (cloud clone, Wuhan, China) according to the manufacturer’s protocol. Data processing was performed with Curve expect software.

Primary bone marrow macrophages (BMMs) were obtained from femurs or tibias of 6–8-week-old C57BL6/J male mice, according to a published method. The cells were cultured and expanded with α-MEM (M0644, Sigma, United States) containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 ng/mL macrophage colony-stimulating factor (M-CSF) for 4 days (Li et al., 2020). For osteoclast differentiation, the adherent BMMs were seeded at 3.125 × 10⁴ cells/cm² and induced with 20 ng/mL M-CSF and 50 ng/mL RANKL for 3–5 days. RAW 264.7 were cultured in the same conditions without M-CSF, and 50 ng/mL RANKL was sufficient for osteoclast differentiation induction. For TRAP staining, cells were fixed with 4% of paraformaldehyde (PFA) for 5–10 min at room temperature and rinsed with water and stained with the acid phosphates, leukocyte (TRAP) kit (387A-1KT, Sigma, St. Louis, United States).

Intracellular acidification was determined by acridine orange (AO) fluorescence method. BMM-derived OCs or RAW264.7 cell-derived OCs were treated with Ad-GFP or Ad-Wnt7b for 12 h. Osteoclasts induced with 50 ng/mL RANKL for 5 days were incubated with 10 μg/mL AO for 15 min at 37°C. Cells were washed twice with PBS and processed for fluorescent microscopy analysis on a NIKON eclipse fluorescence microscope (Compix Inc., Sewickley, PA, United States) at excitation of 485 nm and emission of 520 nm. Images were performed using ImageJ (version 1.47).

To overexpress Wnt7b, RAW264.7 cells were infected with adenovirus encoding mouse Wnt7b or green fluorescent protein (GFP) purchased from Hanbio, Shanghai, China. For viral transfection, cells at the confluence of 80% were incubated with virus overnight, and medium was changed to complete medium. Quantitative PCR (qPCR) and fluorescent imaging were performed to verify transfection efficiency after 24 h transfection. Cells were used for further experiments upon verification.

Total RNA was extracted using TRIzol reagent (Invitrogen Inc., Carlsbad, CA, United States) and was reverse transcribed to cDNA with HiScript^® Q-RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Nanjing, China). Taq Pro Universal SYBR qPCR Master Mix was used for qPCR in CFX96 real-time system. Gene-specific primers are listed in Supplementary Table 1. Relative expression level was calculated by 2^–ΔΔCT and was normalized by β-actin gene.

Cultured cells were washed with PBS and then lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Hudson, NH, United States). Cell extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Primary Abs used are listed in Supplementary Table 2. Horseradish peroxidase (HRP)-conjugated secondary antibodies were probed and developed with ECL solution. Signals were detected and analyzed by Bio-Rad ChemiDoc system (Bio-Rad, Hercules, CA, United States).

Cells were cultured in complete medium for several hours at the confluence of 80%, and they were trypsinized, washed with PBS, and finally fixed in ice-cold 70% ethanol for at least 24 h. Cell cycle analyses followed manufacturer protocols (KGA512, KeyGEN Biotech, Nanjing, China). Cells were washed with PBS twice and incubated with RNase buffer for 30 min at 37°C and stained with PI solution for 30 min on ice. Cells were analyzed by guava easyCyte HT (Millipore, Billerica, MA, United States) and analyzed with software InCyte2.7 (Millipore, Billerica, MA, United States).

EdU staining was performed according to manufacturer’s guideline (C10310-1, Ribo Bio, Guangzhou, China). BMMs were cultured in complete medium at the confluence 50–70% and incubated with complete medium containing EdU at the concentration of 10 μM for 1 h. The labeled cells were subjected to EdU staining and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured by a NIKON eclipse fluorescence microscope (Compix Inc., Sewickley, PA, United States).

Cells were cultured in 96-well plates and changed to 100 μL fresh complete medium before the assays. The CellTiter-Glo kit (G9241, Promega Corporation, Madison, WI, United States) was performed according to the manufacturer’s procedure, and luminescence of each well was detected from the opaque-welled 96-well plates. ATP levels were calculated according to the standard curve generated with 0.1, 1, and 10 μM of ATP and normalized to total DNA amount in each well.

Cell total DNA extraction method was as follows. Cultured cells were washed twice with Hank’s balanced salt solution (HBSS) (with CaCl₂ and MgCl₂, Gibco, Life Technologies Corporation, NY, United States). The plate was emptied before being frozen at −80°C for at least 1 h. About 100 μL or 1 mL of distilled water was added to each well of the plates and placed on an orbital shaker for 1 h at room temperature. One hundred microliters of cell lysates and 100 μL Hoechst 33342 (Thermo Fisher Scientific, Hudson, NH, United States) at 20 μg/mL in TNE buffer were mixed in a black 96-well plate, and fluorescence was detected. DNA amount was calculated according to the standard curve generated before.

Eight 8-week-old female wild-type mice and eight 8-week-old female Wnt7b^LysM mice were randomly divided into two groups to receive sham (four mice in this group) or bilateral OVX surgery (four mice in this group). After anesthesia by isoflurane and 100% oxygen for half hour, the mice in the OVX group were subjected to resection bilateral OVX, and the mice in the sham group underwent some fat tissue close to the ovaries, and all operations above were performed under aseptic conditions. Four weeks later, estrogen-deficiency−induced osteoporosis was successfully established by micro−CT scanning of the femurs of eight mice (four mice in each group) after sacrificed.

Two groups (wild-type and Wnt7b^LysM group) of BMMs were cultured in six-well plates (Corning, Tewksbury MA, United States) at an initial density of 3.125 × 10⁴ cells/cm². Cell proliferation was assessed using a water-soluble tetrazolium salt-WST-8 (Cell Counting Kit-8) according to the manufacturer’s instructions (DOJINDO, Tokyo, Japan). Then, CCK-8 data was read to obtain the absorbance at 450 nm, and the absorbance values of wild type were compared with those of Wnt7b^LysM cells. Cell cycle was quantified by flow cytometry. Cells were stained with propidium iodide (PI) (KGA1015; KeyGEN Bio-TECH, Nanjing, China) following the manufacturer’s directions. Flow cytometry analysis was performed on more than 50,000 events. Data were analyzed with system software (Millipore Guava easyCyte HT, Merck Millipore, Darmstadt, Germany).

One-way ANOVA with post hoc Bonferroni correction was carried out for comparisons of multiple groups. Student’s t-test was performed to determine the statistical significance for two groups. The chi-square test was used to assess the portion of cells in different phases. The level of acceptable statistical significance was set at p < 0.05. Numerical data and histograms were presented as mean ± SD (standard deviations). Results were presented in the presence of at least three independent biological experiments, and for each individual experiment, at least three technical repeats were carried out. All fluorescent microscopic images were processed with Image Pro Plus 6.0 (Media Cybernetics, Rockville, MD, United States).

We previously showed that mice with DMP1-driven Wnt7b overexpression exhibited a high bone mass phenotype. Moreover, the osteogenic differentiation of BMSCs was significantly promoted as expected, which indicated that Wnt7b is a potent activator for osteogenesis (Yu et al., 2020). The reduction in osteoclasts in mutant mice inspired us that Wnt7b might play an important role in osteoclastogenesis in either a cell autonomous or non-autonomous manner.

To test this, we planned to specifically activate Wnt7b in osteoclasts. To this end, we generated RANK-Cre;Rosa26^Wnt7b/+ (hereafter referred to as Wnt7b^RANK) and LysM-Cre;Rosa26^Wnt7b/+ (hereafter referred to as Wnt7b^LysM) conditional knockout mice by crossing Rosa26-Wnt7b with either RANK-Cre or LysM-Cre transgenic mice. At 12 weeks of age, microcomputed tomography (μCT) scanning of the proximal femora was performed. Interestingly, similar to the overexpression of Wnt7b in osteoblasts, the three-dimensional reconstruction of μCT scanning showed a significant increase in bone mass in both trabecular and cortical bone in Wnt7b^RANK and Wnt7b^LysM mice compared to their littermates (Rosa26^Wnt7b, hereafter referred to as wildtype or WT) (Figure 1A). The μCT analysis revealed that the trabecular bone mass (BV/TV) was increased in Wnt7b^RANK and in Wnt7b^LysM compared to WT. The increase in bone mass in mutant mice was due to higher trabecular number (Tb.N.), higher trabecular thickness (Tb.Th), and a decrease in trabecular spacing (Tb.Sp.). The μCT analysis also showed a thicker cortical bone diameter in mutant mice indicated by elevated cortical bone thickness (Ct.Th.) (Figure 1B). Von Kossa staining and Goldner trichrome staining demonstrated deeper staining and thicker bone in mutant mice compared to WT, suggesting more calcium deposition (Figure 1C). Consistent with the lack of change in new bone formation revealed by dynamic histomorphometry in the femur, the serum marker PINP was not impaired (Figures 1D,E). However, we observed a reduced number of TRAP-positive cells in the trabeculae (Figure 1F) and a significantly decreased bone resorption serum marker CTX-1 in mutant mice compared to WT (Figure 1G). The number of TRAP-positive cells in the mandible was diminished in mutant mice (Figure 1H). These data suggest that the specific gain-of-function of Wnt7b in osteoclasts significantly increased bone mass due to a defect in osteoclast differentiation and activity in vivo.

According to the above results, we suggest that Wnt7b has the potential to relieve pathological bone resorption. To further examine this possibility, we generated an OVX mouse model, which exhibited intense bone resorption caused by an estrogen deficit. Six weeks after ovariectomy, μCT analysis revealed that Wnt7b overexpression increased bone mass in both sham and OVX group (Figure 1I). Although we found that the ratio of BV/TV with Wnt7b overexpression showed an increasing trend, the difference was not statistically significant. Due to the lack of changes in the level of reduction in BV/TV, we concluded that Wnt7b achieved a beneficial effect on bone mass, rather than playing a protective effect on OVX-induced osteoporosis in vivo (Figures 1I,J).

In vitro, the BMMs from Wnt7b^LysM mice under RANKL induction formed less osteoclast than those from WT mice as indicated by TRAP staining. Besides, Wnt7b overexpression in RAW264.7 cells and BMMs also abolished osteoclastogenesis (Figures 2A,B). We next performed acridine orange staining on osteoclasts to visualize the intracellular acidification (Figure 2C). The osteoclasts differentiated from BMMs from Wnt7b^LysM mice had less acidification than those from WT mice in the presence of RANKL, which indicated a reduced osteoclast activity. Besides, qPCR experiments indicated that mRNA expression of osteoclast-specific genes, including Nfatc1, C-fos, Trap, Ctsk, Dc-stamp, and Calcr, were significantly repressed in the presence of Wnt7b under osteoclastogenic induction (Figure 2D). Of note, the negatively regulated genes during osteoclast differentiation were also screened by qPCR. Among these genes, Irf8 and Itsn were increased due to Wnt7b overexpression in BMMs (Figure 2E). These results demonstrate that Wnt7b has inhibitory effects on osteoclast differentiation and maturation in vitro.

Previous studies have demonstrated that Wnt ligands such as Wnt3a can modulate the proliferation of BMMs through the canonical Wnt pathway and that β-catenin is essential for osteoclast precursors (Hamamura et al., 2014; Weivoda et al., 2016). Therefore, we next examined the cell viability of BMMs in the presence of Wnt7b using CCK-8 cell counting experiments. As a result, we detected a reduction in the viability of BMMs derived from Wnt7b^LysM mice indicated by CCK-8 experiment (Figure 3A). Next, flow cytometry was used to analyze the phases of the cell cycle. Surprisingly, there was no remarkable difference between Wnt7b overexpression and control in terms of cell cycle (Figure 3B). EdU staining of cultured BMMs from either WT or Wnt7b^LysM mice also confirmed the flow cytometry results (Figure 3C). Meanwhile, the cell-cycle-related genes, such as Cdk2, Cdk4, P21, P27, and P53, did not change when enforced Wnt7b expression in BMMs (Figure 3D). Next, mRNA expression of Wnt receptors, such as Reck, Gpr124, and Lrp5/6, and Wnt downstream genes, such as Axin2 and Gsk3β, Lef1, Ror2, and Pcna, were examined by qPCR (Figure 3E). We found that the mRNA expression of majority of Wnt receptors showed no significant changes between WT and Wnt7b^LysM, and genes related to β-catenin-dependent Wnt signal did not activate in response to Wnt7b in BMMs. Furthermore, the decreased transcriptional level of Lrp6 and Axin2 in the presence of Wnt7b indicated that the canonical Wnt signal might be suppressed in BMMs with Wnt7b overexpression. We further explored the protein levels of β-catenin in cell lysates by Western blotting and found that β-catenin was obviously decreased in the BMMs derived from Wnt7b^LysM mice compared to those from the WT mice (Figure 3F). In addition, we treated the BMMs with Wnt7b conditional medium and detected that the protein level of β-catenin in BMMs was also reduced in response to Wnt7b in a time-dependent manner (Figure 3G). Data indicated that the β-catenin-dependent pathway was suppressed in the presence of Wnt7b in BMMs.

Several signaling pathways, such as nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin (mTOR), are involved in osteoclast differentiation (Liu and Zhang, 2015). We screened whether Wnt7b inhibited the key activation step of several signaling pathways induced by RANKL. In the canonical NF-κB pathway, phosphorylation and nuclear translocation of p65 plays a critical role during RANKL-induced NF-κB activation. Western blotting analysis revealed that Wnt7b inhibited or at least delayed RANKL-induced p65 phosphorylation (Figure 4A) and the non-canonical NF-κb activation indicated by Ikk-a protein level (Abu-Amer, 2013). The RANKL-induced p38 and JNK phosphorylation also was not affected by Wnt7b overexpression (Figure 4B). Since AKT, a critical serine-threonine kinase, is reportedly mediating diverse signaling pathway downstream of mTORC2, we tested the phosphorylation of AKT at Ser473. We observed that Wnt7b significantly abolished AKT phosphorylation at Ser473, whereas activation of PKC and S6K1 was not significantly impaired with RANKL treatment for 15 min (Figure 4C).

Next, we investigated whether SC79, an AKT activator, rescued osteoclast formation suppressed by Wnt7b (Zhang et al., 2016). We pretreated BMMs with SC79 or dimethyl sulfoxide (DMSO) for 24 h before inducing them with RANKL for additional 5 days. The protein expression of osteoclast-differentiation-related genes were upregulated after SC79 treatment (Figure 4D). Of note, BMMs derived from Wnt7b^Lysm mice previously failed to form TRAP-positive cells, whereas the number of osteoclasts increased with SC79 treatment in the presence or absence of Wnt7b (Figure 4E). The mRNA expressions of osteoclast-differentiation-related genes were rescued after SC79 treatment (Figure 4F). Thus, we concluded that Wnt7b abolished osteoclast formation by inhibiting AKT phosphorylation.

Recent studies have shown that the cell metabolic status could regulate signal pathway activation and contribute to the regulation of cell differentiation (Lecka-Czernik and Rosen, 2015). Moreover, ATP generated from anerobic glycolysis has been shown to fuel the PI3K-AKT pathway to support the Th17 cell response (Xu et al., 2021a,b). Previous studies have revealed that glycolysis and OXPHOS were both enhanced during osteoclast differentiation. Meanwhile, glucose consumption and the expression of glucose transporters (GLUTs) were elevated as the osteoclast differentiated (Li et al., 2020). Therefore, we examined ATP levels, glucose consumption, and lactate production to determine the metabolic condition of BMMs derived from Wnt7b^Lysm mice compared to those from WT mice (Figures 5A–C). The ATP levels were unchanged; however, glucose consumption and lactate production dropped significantly. Then, we screened the transcriptional level of GLUTs family in BMMs. Glut1, a member of glucose transporter family, identified as the predominant GLUTs in BMMs, showed no significant change in the presence of Wnt7b, while Glut3 presented a mild increase (Figure 5D). Thus, GLUT family was dispensable for the inhibition of glucose utilization caused by Wnt7b.

Furthermore, the transcriptional levels of the metabolic enzymes were examined by qPCR. The data demonstrated that most of the glycolysis-related genes were steadily expressed, whereas the expression of Pgk1 and Pdha was dramatically decreased in the presence of Wnt7b (Figure 5E). Some published studies revealed that Pgk1 was a central enzyme in glycolysis process, which generated a molecular of ATP. The mRNA expression of Pgk1 decreased in BMMs with Wnt7b forced expression suggesting that Wnt7b impaired glycolysis in BMMs with Wnt7b overexpression. Meanwhile, we found that Wnt7b forced expression led to slight upregulation of Pdk2 and downregulation of Pdha, which controlled the turnover of TCA cycle. These data suggested that the glucose metabolic process was rewired by Wnt7b in BMMs.

Next, we assayed the metabolites in BMMs with or without Wnt7b overexpression for 24 h before RANKL treatment (Figure 5F). The metabolites in the TCA cycle were elevated in RANKL-stimulated BMMs compared to the unstimulated BMMs, which indicated an increasing glucose usage in TCA during osteoclastogenesis. Importantly, with RANKL inducement, the content of citrate was significantly reduced in the presence of Wnt7b. Therefore, we investigated whether resupplemented citrate into the medium rescued osteoclast formation, which was suppressed by Wnt7b. To this end, we first tested the intracellular amount of citrate in BMMs after citrate compensation. After treatment with additional citrate in the medium, the intracellular citrate content was increased in BMMs (Figure 5G), as were the protein levels of AKT and the osteoclast differentiation markers PU.1 and C-fos (Figure 5H). Moreover, the TRAP staining also demonstrated that the BMMs derived from Wnt7b^LysM mice form more osteoclasts in the presence of 10 mM citrate (Figure 5I), which indicated that citrate compensation rescued osteoclastogenesis. Therefore, we concluded that Wnt7b impaired AKT phosphorylation and rewired the glucose metabolic process in BMMs during osteoclastogenesis.