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Introduction

Adenosine kinase (ADK) is the key metabolic regulator of the purine ribonucleoside adenosine and is essential for intracellular adenosine metabolic clearance via phosphorylating adenosine to AMP based on its low Km(1). ADK is expressed in most vital organs that require metabolic adenosine clearance, in particular the liver and placenta (2). ADK controls specific organ functions via a combination of adenosine receptor-dependent and -independent mechanisms (1). Changes in adenosine homeostasis may activate the four adenosine-associated G-protein-coupled receptors, alter energy metabolism and regulate DNA methylation (3). A number of studies have demonstrated that ADK serves important roles in several diseases, including epilepsy (4), traumatic brain injury (5), stroke (6), diabetes (7) and cancer (8).

Bone is a highly dynamic organ that is constantly undergoing remodeling by means of bone formation and resorption in the adult skeleton (9). Previous studies have investigated the role of adenosine and its four G protein-coupled receptors in the maintenance of bone homeostasis in vivo and in vitro, particularly adenosines association with osteoclastogenesis and bone resorption in rodents and humans (10). Previous reports suggest that activating adenosine A1 receptor (A1R) and adenosine A2B receptor (A2BR) promotes osteoclastogenesis; however, adenosine A2A receptor (A2AR) activation has the opposite effect (11–14) and adenosine A3 receptor (A3R) activation indirectly inhibits osteoclastogenesis due to its anti-inflammatory effect (15).

ADK deficiency results in the accumulation of intracellular adenosine and a decrease in AMP, ADP and ATP, which in turn leads to disordered purinergic signaling (1). A recent clinical investigation based on next generation sequencing revealed that patients deficient in ADK with intellectual disabilities also presented with short stature, frontal bossing and slender hands and fingers (16). In a mouse model, genetic deletion of ADK resulted in mice succumbing within 14 days of birth whereas mice with ADK deficiency exhibited reduced body weight and length (17). However, the function of ADK in bone metabolism is still unknown.

Osteoclasts are the primary bone-resorbing cells and are thought to be derived from the monocyte-macrophage lineage (18). In the present study, a mouse model with ADK knockout in myeloid monocyte cells (ADKMMC-KO) was established via Cre/LoxP site-specific recombination. The effect of ADK on bone metabolism was assessed by gross observation and bone histomorphometric analysis in vivo to ascertain whether there were changes in bone homeostasis. In addition, osteoclast differentiation and bone resorption were examined in vitro by genetic deletion and pharmacologic inhibition of ADK in osteoclasts.

Materials and methods

Animals

All animal experiments in this study were approved and supervised by the Institutional Animal Care and Use Committee (IACUC No. 2014027) of Guangdong Laboratory Animals Monitoring Institute (Guangzhou, China). ADKflox/flox (C57BL/6 background) mice (hereafter referred to as ADKWT) provided by Professor Yuqing Huo (Vascular Biology Center, Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA, USA) (19) were mated to lysozyme 2-Cre mice (lysMcre; stock no. 004781; Jackson Laboratory, Farmington, CT, USA), in which the Cre recombinase gene is knocked into the lysozyme 2 locus and is specifically expressed in myeloid monocyte cells (20). Mice with ADK-deficient myeloid monocyte cells (ADKMMC-KO) were generated by mating ADKflox/floxxlysMcre mice with ADKflox/flox mice. All mice were provided with water and a standard chow diet ad libitum. Mice were maintained in a specific pathogen-free facility with a 12 h light/dark cycle at 23±2°C and 50±10% relative humidity.

For the in vivo experiment, 4-month-old male ADKMMC-KO (23–25 g; n=10) and ADKWT (27–29 g; n=10) mice were used for gross apperance and bone histomorphometric analysis, with ADKWT as the control. For the in vitro experiment, 2-month-old male ADKMMC-KO (21–23 g; n=10) and ADKWT mice (25–27 g; n=10) were used for osteoclastogenesis and bone resorption analysis. In addition, 2-month-old male ADKWT mice (25–27 g; n=20) were assigned to either an ABT-702-treated group (n=10) or a vehicle control group (n=10) for in vitro osteoclastogenesis and bone resorption analysis.

Cell culture

Murine osteoclasts were generated from bone marrow cells as previously described (21). Briefly, following CO2 euthanasia, primary ADKMMC-KO or ADKWT mouse bone marrow cells were isolated by flushing the tibia and femur of 2-month-old male mice with α-modified Eagle's medium (α-MEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were subsequently centrifuged (1,000 × g) at 4°C for 5 min and incubated in α-MEM containing 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, Inc.), penicillin-streptomycin (100 U/ml) and 1% (w/v) glutamine (Sigma Aldrich; Merck KGaA, Darmstadt, Germany) overnight at 37°C in a humidified atmosphere containing 5% CO2. To generate bone marrow-derived macrophages, non-adherent cells were collected, plated onto 24-well plates (1×106 cells/well) and cultured at 37°C in a humidified atmosphere containing 5% CO2 with 25 ng/ml macrophage colony-stimulating factor (M-CSF; R&D Systems, Inc., Minneapolis, MN, USA) for 3 days in α-MEM. Cells were then used for osteoclast differentiation or ADK western blot analysis. To generate osteoclasts, bone marrow-derived macrophages were incubated at 37°C in a humidified atmosphere containing 5% CO2 with α-MEM containing 25 ng/ml M-CSF and 30 ng/ml receptor activator of nuclear factor kappa-B ligand (RANKL; R&D Systems, Inc.) for 5 days. To evaluate the effect of ADK inhibition on osteoclast differentiation, ABT-702 dihydrochloride (Tocris; Bio-Techne, Minneapolis, MN, USA), a potent non-nucleoside ADK inhibitor, was pre-dissolved in 5% EtOH and the solution was diluted with osteoclast differentiation medium as described for cell culture. All assays were performed in triplicate.

Western blotting

Total protein was extracted from bone marrow-derived macrophages using radioimmunoprecipitation assay buffer (Sigma Aldrich; Merck KGaA) according to the manufacturer's protocol. Protein was quantified using a bicinchoninic acid assay and 20 µg samples were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% (w/v) non-fat dry milk in TBST buffer for 1 h at 4°C. Rabbit monoclonal antibodies against ADK (Sigma Aldrich; Merck KGaA; cat no. SAB2701969; 1:1,000) and GAPDH (Sigma Aldrich; Merck KGaA; cat. no. G9545, 1:2,500) were used to assess ADK protein levels in cultured cell lysates. The membranes were incubated with the primary antibodies in 1% bovine serum albumin (Sigma Aldrich; Merck KGaA) overnight at 4°C and washed three times with TBST. Subsequently, membranes were incubated with horseradish peroxidase-conjugated secondary goat anti rabbit antibody (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. 31460; 1:2,000) in non-fat dry milk for 1 h at room temperature and washed three times with TBST. Membranes were visualized using West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.). Bands were quantified using Image Lab 5.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Tartrate-resistant acid phosphatase (TRAP) activity assay

TRAP staining was performed using an Acid Phosphatase Leukocyte (TRAP) kit (Sigma Aldrich; Merck KGaA) according to the manufacturer's protocol. Briefly, osteoclasts differentiated from bone marrow cells were fixed in 37% formaldehyde for 30 sec at 4°C and rinsed thoroughly in deionized water. TRAP staining solution was mixed and preheated to 37°C and the fixed cells were added and incubated for 1 h at 37°C. Cells were counterstained with hematoxylin for 30 sec at room temperature and the number of osteoclasts (defined as multinuclear TRAP-positive cells) per well was counted under a bright field inverted microscope (magnification, ×200; DMI3000; Leica Microsystems GmbH, Wetzlar, Germany).

Bone resorption assay and assessments

A bone resorption assay was performed as previously described (22). Briefly, bone marrow-derived macrophages (1×106 cells/well) were seeded in Osteo-Assay Surface 24-well plates (Sigma Aldrich; Merck KGaA); osteoclastic differentiation and culture were performed as above. Plates were examined under a bright field inverted microscope (magnification, ×10) and images of 10 random fields/well were captured. The percentage and number of resorption areas were calculated using with ImageJ software 1.50i (National Institutes of Health, Bethesda, MD, USA).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from cultured cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) and RT was performed using the PrimeScript™ RT reagent kit (Takara Bio, Inc., Otsu, Japan) at 37°C for 30 min and 85°C for 1 min according to the manufacturer's protocol. All primers were provided by Invitrogen (Thermo Fisher Scientific, Inc.) and were as follows: TRAP, forward 5′-ACCATTGTTAGCCACATACG-3′ and reverse 5′-GTGAAACCGCAAGTAGCC-3; matrix metalloproteinase-9 (MMP-9), forward 5′-AGTTTGGTGTCGCGGAGCAC-3′ and reverse 5′-TACATGAGCGCTTCCGGCAC-3′; nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), forward 5′-GGGTCAGTGTGACCGAAGAT-3′ and reverse 5′-GGAAGTCAGAAGTGGGTGGA-3′; proto-oncogene tyrosine-protein kinase Src (c-Src), forward 5′-CCAGGCTGAGGAGTGGTACT-3′ and reverse 5′-CAGCTTGCGGATCTTGTAGT-3′; FBJ osteosarcoma oncogene (c-FOS), forward 5′-CCAGTCAAGAGCATCAGCAA-3′ and reverse 5′-AAGTAGTGCAGCCCGGAGTA-3′; Cathepsin K (CtsK), forward 5′-GAAGAAGACTCACCAGAAGCAG-3′ and reverse 5′-TCCAGGTTATGGGCAGAGATT-3′; calcitonin receptor (CTR), forward 5′-CCTGACAGCAACCGAACC-3′ and reverse 5′-GCAACCAAAGCAGCAATC-3′; GAPDH, forward 5′-TGTAGACCATGTAGTTGAGGTCA-3′ and reverse 5′-AGGTCGGTGTGAACGGATTTG-3′. qPCR reactions were performed as follows: 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec, using an ABI-7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) in a total volume of 25 µl containing 2 µM primers and 12.5 µl of the SYBR® Premix Ex Taq™ (Takara Bio, Inc.). Results were quantified using the 2−ΔΔCq method (23).

Bone histomorphometric analysis

The proximal tibia metaphysis and tibia shaft were fixed at 4°C for 48 h in 4% paraformaldehyde and decalcified in 10% EDTA for 3–4 weeks. Tibias were subsequently embedded in paraffin and cut into 4-µm sections. Following deparaffinization and rehydration, sections were stained with hematoxylin and eosin (H&E) for 1 min at room temperature. Terminology recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (24) and the BIOQUANT OSTEO 2009 morphometric measuring system (Bioquant Image Analysis Corporation, Nashville, TN, USA) were used in the present study. The proximal tibia metaphysis histomorphometric perimeters were determined by measuring the areas under 1–3 µm from the growth plate excluding the primary spongiosa and trabeculae connected to the cortical bone. The lower end of the tibia was separated from the fibula and prepared for tibia shaft transection and histomorphometric perimeters were determined.

Statistical analysis

Two-tailed unpaired t-tests with Welch's correction were used for comparisons between the two groups, using GraphPad Prism 6.01 (GraphPad Software, Inc., La Jolla, CA, USA). Data are presented as the mean + standard error of the mean of three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

Results

Spontaneous dwarfism in ADKMMC-KO mice

Primordial dwarfism was observed in ADKMMC-KO mice. Compared with male ADKWT mice, the appearance of ADKMMC-KO mice was not markedly different, however the mice appeared markedly smaller at 4 months old (Fig. 1A). Measurements revealed that the body weight and nasoanal length of ADKMMC-KO mice was significantly decreased compared with ADKWT mice (P<0.0001; Fig. 1B).

Figure 1 Longitudinal growth retardation in ADKMMC-KO mice. (A) Gross appearance of 4-month male ADKWT and ADKMMC-KO mice. Quantitative analysis of (B) body weight and nasoanal length, (C) tibia and femur length and (D) tibia and femur weight in 4-month-old male ADKWT and ADKMMC-KO mice. n=10 per group. ****P<0.0001 vs. ADKWT mice. ADK, adenosine kinase; ADKWT, ADK wild type; ADKMMC-KO, ADK knockout in myeloid monocyte cells.

To investigate whether the dwarfism observed in ADKMMC-KO mice was associated with skeletal growth, the tibia and femur were assessed. The results demonstrated that the tibia and femur lengths were significantly shorter in ADKMMC-KO mice compared with ADKWT mice (P<0.0001; Fig. 1C). Furthermore, it was demonstrated that the tibia and femur weigh significantly less in ADKMMC-KO mice compared with ADKWT mice (P<0.0001; Fig. 1D). These results suggest that conditional ADK knockout in myeloid monocyte cells causes skeletal dysostosis in mice.

Abnormal bone formation in ADKMMC-KO mice

To further investigate the causes of skeletal dysostosis in ADKMMC-KO mice, bone samples underwent histomorphometric analysis. In the proximal tibia metaphysis, fewer bone trabeculae and a smaller volume of trabecular bones was observed in ADKMMC-KO mice compared with ADKWT mice (Fig. 2A). Quantitative analysis of bone trabecular parameters revealed that the bone area (%Tb.Ar; P<0.01), trabecular thickness (Tb.Th; P<0.05) and trabecular number (Tb.N; P<0.05) were significantly decreased in ADKMMC-KO mice compared with ADKWT mice (Fig. 2B). Furthermore, trabecular separation (Tb.Sp; P<0.05) was significantly increased in ADKMMC-KO mice compared with ADKWT mice (Fig. 2B). Analysis of osteoclastic parameters revealed that the percentage of trabecular osteoclast surface to bone surface (%Oc.S/BS; P<0.01) and ratio of osteoclast number to bone surface (N.Oc/BS; P<0.01) were significantly increased in ADKMMC-KO mice compared with ADKWT mice (Fig. 2B). However, the adherent perimeter of osteoblasts (%Ob.S/BS; P<0.01) and number of osteoblasts per unit bone surface (N.Ob/BS; P<0.01), were significantly lower in in ADKMMC-KO mice compared with ADKWT mice (Fig. 2B).

Figure 2 Histological analysis of the tibia from ADKMMC-KO mice. (A) H&E staining of the proximal tibia metaphysis of ADKMMC-KO and ADKWT mice. (B) Bone histomorphometric analysis of %Tb.Ar, Tb.Th, Tb.N, Tb.Sp, %Oc.S/BS, N.Oc/BS, %Ob.S/BS and N.Ob/BS for ADKWT and ADKMMC-KO mice. (C) H&E staining of the tibia shaft from ADKMMC-KO and ADKWT mice. (D) Bone histomorphometric analysis of %Ct.Ar and %Ma.Ar in the middle part of tibia shaft from ADKWT and ADKMMC-KO mice. n=10 per group. Scale bar=200 µm. *P<0.005 and **P<0.01 vs. ADKWT. ADK, adenosine kinase; ADKWT, ADK wild type; ADKMMC-KO, ADK knockout in myeloid monocyte cells; H&E, hematoxylin and eosin; %Tb.Ar, percentage of bone area; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; %Oc.S/BS, percentage of adherent perimeter cells; N.Oc/BS, number of osteoclasts per unit bone surface; %Ob.S/BS, percentage of adherent perimeter osteoblasts; N.Ob/OS, number of osteoblasts per unit bone surface; %Ct.Ar, percentage of cortical bone area; %Ma.Ar, bone marrow cavity area.

The tibial shaft of in ADKMMC-KO mice was also examined and, compared with ADKWT mice, the cortical bone was markedly reduced and an expansive bone marrow cavity was observed (Fig. 2C). Quantitative analysis revealed that the percentage of cortical bone area was significantly decreased (%Ct.Ar, P<0.01) and the bone marrow cavity area was significantly increased (%Ma.Ar; P<0.01) in ADKMMC-KO mice (Fig. 2D). These results suggest that conditional ADK knockout in myeloid monocyte cells causes defective bone formation in ADKMMC-KO mice.

Inhibition of ADK promotes osteoclastogenesis

To further understand whether spontaneous osteoporosis in ADKMMC-KO mice is due to ADK inhibition, bone marrow macrophages (BMMs) were harvested from 2-month-old male ADKWT and ADKMMC-KO mice. Western blotting revealed that the expression of ADK protein was significantly decreased in ADKMMC-KO compared with ADKWT mice (Fig. 3A, P<0.001). BMMs were subsequently exposed to M-CSF and RANKL for 8 days to induce osteoclastic differentiation. A TRAP assay was performed to reveal TRAP-positive cells and the typical multinucleated characteristics of in vitro differentiated osteoclasts were observed (Fig. 3B). Notably, larger TRAP-positive osteoclasts were observed in ADKMMC-KO cultures compared with those from ADKWT mice (Fig. 3B). It was also demonstrated that BMMs derived from ADKMMC-KO mice formed significantly more osteoclasts compared with those derived from ADKWT mice (P<0.01; Fig. 3B). Factors associated with osteoclast formation were evaluated using RT-qPCR and the results indicated that relative levels of TRAP (P<0.01) and NFATc1 (P<0.05) mRNA were significantly increased in ADKMMC-KO osteoclasts compared with ADKWT osteoclasts (Fig. 3C); however, no significant difference was observed in c-FOS expression (Fig. 3C).

Figure 3 ADK knockdown enhances osteoclastogenesis and the expression of associated genes. (A) Expression of ADK protein in BMMs from 2-month male ADKWT and ADKMMC-KO mice. (B) TRAP staining of osteoclasts generated from BMMs of ADKWT and ADKMMC-KO mice. (C) Relative TRAP, NFATc1 and c-FOS mRNA in osteoclasts generated from BMMs of ADKWT and ADKMMC-KO mice. (D) TRAP staining analysis of osteoclasts generated from BMMs of ADKWT mice treated with 100 nM ABT-702. (E) Relative TRAP, NFATc1 and c-FOS mRNA levels in osteoclasts generated from BMMs of ADKWT mice treated with 100 nM ABT-702 Scale bar=500 µm. *P<0.05, **P<0.01 and ***P<0.01 vs. control. ADK, adenosine kinase; BMMs, bone marrow macrophages; ADKWT, ADK wild type; ADKMMC-KO, ADK knockout in myeloid monocyte cells; TRAP, tartrate-resistant acid phosphatase; NFATc1, nuclear factor of activated T-cells, cytoplasmic 1; Control, untreated ADKWT mice.

To further verify the role of ADK in osteoclast formation, the ADK inhibitor ABT-702 was applied to BMMs from ADKWT mice to induce osteoclastic differentiation (Fig. 3D). Treatment with 100 nM ABT-702 significantly induced TRAP-positive osteoclast formation compared with untreated ADKWT BMMs (P<0.01; Fig. 3D). The expression of TRAP (P<0.05) and NFATc1 (P<0.05) mRNA was significantly increased by treatment with 100 nM ABT0702 compared with untreated ADKWT BMMs, similar to ADKMMC-KO cultures (Fig. 3E). These results suggest that ADK inhibition induces osteoclast formation.

ADK inhibition increases osteoclastic resorption activity

To investigate whether ADK inhibition affects osteoclastic resorption function, BMMs derived from 2-month-old male ADKWT and ADKMMC-KO mice were incubated with M-CSF and RANKL. Following 7 days of culture, the resorption area of osteoclasts induced from ADKMMC-KO BMMs was significantly greater than that of osteoclasts induced from ADKWT BMMs (P<0.05; Fig. 4A). Similarly, the number of resorption areas per well was significantly greater in the ADKMMC-KO group compared with the ADKWT GROUP (P<0.05; Fig. 4A). Factors associated with osteoclast formation were further evaluated in these cultures using RT-qPCR the results demonstrated that relative levels of MMP9 (P<0.01), CtsK (P<0.001) and CTR (P<0.01) mRNA were significantly increased in ADKMMC-KO osteoclasts compared with ADKWT osteoclasts (Fig. 4B). However, no significant difference was observed in c-Src expression (Fig. 4B).

Figure 4 ADK knockdown enhances osteoclastic resorption activity and the expression of associated genes. (A) The resorption activity of osteoclasts generated from BMMs of ADKWT and ADKMMC-KO mice. (B) Relative MMP9, CtsK, c-Src and CTR mRNA expression in osteoclasts generated from BMMs of ADKWT and ADKMMC-KO mice. (C) The resorption activity of osteoclasts generated from BMMs of ADKWT treated with 100 nM ABT-702. (D) Relative MMP9, CtsK, c-Src and CTR mRNA expression in osteoclasts generated from BMMs of ADKWT mice treated with 100 nM ABT-702. Scale bar=200 µm. *P<0.005, **P<0.01 and ***P<0.001 vs. control. ADK, adenosine kinase; BMMs, bone marrow macrophages; ADKWT, ADK wild type; ADKMMC-KO, ADK knockout in myeloid monocyte cells; MMP9, matrix metalloproteinase 9; c-FOS, FBJ osteosarcoma oncogene; CtsK, Cathepsin K; c-Src, proto-oncogene tyrosine-protein kinase Src; CTR, calcitonin receptor; Control, untreated ADKWT mice.

BMMs derived from ADKWT mice were also treated with 100 nM ABT-702 to further evaluate the effect of ADK inhibition on osteoclastic resorption. The relative resorption area (P<0.001) and number of resorption areas per well (P<0.01) was significantly increased in cells treated with ABT-702 compared with untreated BMMs derived from ADKWT mice (Fig. 4C). The relative expression of MMP9 (P<0.01) and CtsK (P<0.05) mRNA was significantly increased in ABT-702 cultures compared with untreated BMMs derived from ADKWT mice (Fig. 4D). However, no significant difference was observed in c-Src and CTR levels (Fig. 4D). These results suggest that ADK inhibition may promote osteoclastic resorption activity

Discussion

In the present study, a mouse model with ADK deficient myeloid monocyte cells (ADKMMC-KO) was established by Cre/LoxP site-specific recombination. As a result of this conditional ADK knockout, spontaneous dwarfism associated with decreased bone mass was observed in adult mice. Further in vitro investigations based on genetic and pharmaceutical ADK disruption in bone marrow macrophages revealed that ADK inhibition significantly increases osteoclastogenesis and bone resorption in mouse BMMs. Collectively, these results suggest that ADK may be a potential target for the treatment of metabolic bone diseases associated with osteoclast.

Previous clinical studies have reported that human patients with global ADK deficiency due to homozygous point mutations exhibit developmental delay, including short stature, frontal bossing and slender hands and fingers (16,25). A study involving homozygous disruption of the ADK gene in mice also reported reduced body size and weight within a few days of birth (1). In the present study, ADKMMC-KO mice were characterized by spontaneous dwarfism, including reduced body weight, nasoanal length, and weight and length of tibia and femur. Histomorphometric analysis revealed significantly decreased bone mass in proximal tibia metaphysis and the middle of the tibial shaft in ADKMMC-KO mice. Combined with previous reports, these results indicate that ADK may serve an important role in bone metabolism homeostasis.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow (26). lysMcre mice have been used extensively to investigate osteoclast function in previous studies (27–29). In the present study, osteoclastic changes in ADKMMC-KO mice were assessed in vivo using histomorphometric analysis and in vitro using a BMM cell culture. The results revealed increased osteoclastic parameters, including osteoclastogenesis and bone resorption, associated with ADK deficiency. Histomorphometric analysis also demonstrated that the function of osteoblasts derived from pluripotent mesenchymal stem cells was inhibited in ADKMMC-KO mice, suggesting that purinergic signaling from ADK-deficient activated osteoclasts may affect osteoblasts by interacting with adenosine receptors (10). Furthermore, the exosomes derived from activated osteoclasts may also inhibit osteoblast activity (30).

ADK converts intracellular adenosine to AMP; ADK deficiency or dysfunction results in the accumulation of intracellular adenosine (1). Adenosine may be transported to the extracellular environment by type 1 equilibrative nucleoside transporter (ENT1) (31); as such, the ADK-deficiency-induced accumulation of intracellular adenosine may result in adenosine being transported to the extracellular environment by ENT to interact with adenosine receptors, including A1R (32,33) and A2BR (34,35), to promote osteoclast differentiation. However, mice lacking ENT1 have been reported to have reduced weight and lower bone density in the spine and femur associated with increased osteoclast activity (36). This suggests that intracellular adenosine may also participate in the functional regulation of osteoclasts via a receptor independent pathway, although this requires further investigation.

Metabolic analysis of liver tissue homogenates from homozygous ADK deficient mice revealed a 40% decrease in AMP and ADP and a 35% decrease in ATP (17). A previous study utilizing osteoclast-specific conditional knockout Tfam mice, in which ATP production is inhibited in osteoclasts, reported reduced growth and increased bone-resorption (37). An in vitro study demonstrated that ATP inhibits the differentiation of osteoclast-like cells from RANKL-induced RAW cells (38). A number of studies have reported that metformin is able to effectively improve osteoporosis in patients with long-term type 2 diabetes mellitus by inhibiting osteoclast differentiation (39–41). The mechanism by this is achieved is associated with the activation of AMP-activated protein kinase (AMPK), a 'metabolic master switch' downstream of ADK (42–44). It was therefore speculated that prolonged ADK deficiency in myeloid monocyte cells may result in disordered purinergic signaling, which may contribute to bone growth retardation and excessive osteoclast differentiation and activation in ADKMMC-KO mice.

In summary, the results of the present study suggest that ADK deficient myeloid monocyte cells contribute to reduced bone formation in mice and this is associated with osteoclastogenesis and bone resorption in ADKMMC-KO mice.

Acknowledgments

The authors would like to thank Professor Yuqing Huo for providing the ADK conditional knockout mice. The present study was supported by the Guangdong Provincial Science & Technology Project (grant nos. 2013B060300025, 2015A030303006 and 2016A030313790 to G.L.) and the Natural Science Foundation of China (grant nos. 31702074 to G.L. and 31472061 to Y.Z.).

Notes

[1] Competing interests

The authors declare that they have no competing interests.

References