Intracellular and extracellular ATP coordinately regulate the inverse correlation between osteoclast survival and bone resorption

Abstract

Osteoclasts, highly differentiated bone-resorbing cells of hematopoietic origin, have two conflicting tendencies: a lower capacity to survive and a higher capacity to execute energy-consuming activities such as bone resorption. Here, we report that when compared with their precursors, mature mitochondria-rich osteoclasts have lower levels of intracellular ATP, which is associated with receptor activator of nuclear factor κ-B ligand (RANKL)-induced Bcl-x(L) down-regulation. Severe ATP depletion, caused by disrupting mitochondrial transcription factor A (Tfam) gene, leads to increased bone-resorbing activity despite accelerated apoptosis. Although AMP-activated protein kinase (AMPK) activation by ATP depletion is not involved in the regulation of osteoclast function, the release of ATP from intracellular stores negatively regulates bone-resorbing activity through an autocrine/paracrine feedback loop by altering cytoskeletal structures. Furthermore, osteoclasts derived from aged mice exhibit reduced mitochondrial DNA (mtDNA) and intracellular ATP levels with increased bone-resorbing activity, implicating the possible involvement of age-related mitochondrial dysfunction in osteoporosis. Thus, our study provides evidence for a mechanism underlying the control of cellular functions by reciprocal changes in intracellular and extracellular ATP, which regulate the negative correlation between osteoclast survival and bone resorption.

Figures

FIGURE 1.

Mature osteoclasts display lower intracellular ATP levels associated with decreased expression of Bcl-xL.A, mitochondrial protein content in BMMs, osteoclasts, and osteoblasts. The protein content of the mitochondrial fractions was expressed as the percentage of total protein of the corresponding whole-cell extract. Mitochondrial protein content in mature osteoclasts was higher than that in BMMs or osteoblasts. B, intracellular ATP levels in BMMs, osteoclasts, and osteoblasts. Intracellular ATP levels in osteoclasts was markedly lower than not only osteoblasts, but also BMMs. C, effect of adenovirus-mediated expression of GFP (AxGFP; control), constitutively active MEK (AxMEKCA), Bcl-2 (AxBcl-2), Bcl-xL (AxBcl-xL), or kinase-dead Csk (AxCskKD) on intracellular ATP levels in osteoclasts. Intracellular ATP levels in osteoclasts increased dramatically upon Bcl-xL expression. D and E, real-time PCR analysis of Bcl-xL mRNA level (D) and expression of Bcl-xL protein (E) in BMMs and osteoclasts. Both mRNA and protein expression levels of Bcl-xL in mature osteoclasts were markedly lower than in BMMs. F, effect of RANKL stimulation on expression level of Bcl-xL in BMMs. Bcl-xL expression level in BMMs was gradually reduced by RANKL stimulation. G, effect of retrovirus-mediated expression of small hairpin RNA targeting Bcl-xL (RxshBcl-xL) or Bcl-xL (RxBcl-xL) on NFATc1 protein levels in BMMs stimulated with RANKL. Increase and decrease in the expression level of NFATc1 were observed in shBcl-xL and Bcl-xL transfectants, respectively. RxCtrl indicates an empty retroviral control vector. H, osteoclast differentiation from BMMs infected with RxCtrl, RxshBcl-xL, or RxBcl-xL in response to RANKL and M-CSF. Cells were stained to evaluate TRAP-positive multinucleated osteoclast formation. Scale bar: 100 μm. *, p < 0.05, **, p < 0.01.

FIGURE 2.

Generation and skeletal analysis of Tfam cKO mice.A, expression of Tfam, actin, and Cre proteins in osteoblasts and osteoclasts derived from Tfam cKO mice and their normal Tfamflox/flox littermates. Tfam was markedly reduced in osteoclasts derived from Tfam cKO mice. B, representative photographs and body weight of male Tfam cKO mice and their normal Tfamflox/flox littermates at 8 weeks of age. C and D, representative radiography images of the femur (C) and distal femur microcomputed tomography (D) of male Tfam cKO mice and their normal Tfamflox/flox littermates at 8 weeks of age. Scale bar: 1000 μm. E, histological analysis of proximal tibia of male Tfam cKO mice and their normal Tfamflox/flox littermates at 8 weeks of age (TRAP/hematoxylin staining). The number of TRAP-positive cells is markedly decreased in Tfam cKO mice. Scale bar: 100 μm. F, bone volume and parameters for osteoclastic bone resorption in the bone morphometric analysis of male Tfam cKO mice and their normal Tfamflox/flox littermates at 8 weeks of age (n = 5 for each genotype). Eroded surface per osteoclast (ES/Oc.N) was increased by Tfam ablation. NS, not significant; AU, arbitrary units. G, parameters for osteoblastic bone formation in the bone morphometric analysis of male Tfam cKO mice and their normal Tfamflox/flox littermates at 8 weeks of age (n = 5 for each genotype). *, p < 0.05, **, p < 0.01 versus normal Tfamflox/flox littermates.

FIGURE 3.

Tfam cKO osteoclasts show increased bone-resorbing activity despite accelerated apoptosis.A, electron microscopy of osteoclasts derived from Tfamflox/flox and Tfam cKO mice. Mitochondria in Tfam cKO osteoclasts appeared to display abnormal internal compartmentalization. Higher magnification images of the framed areas are shown in the right panels. Swollen and disorganized cristae were observed in Tfam cKO osteoclasts. Scale bar: 100 nm. B, mitochondrial protein content in Tfamflox/flox and Tfam cKO osteoclasts. Mitochondrial protein content was slightly but significantly reduced by Tfam deficiency. C, mtDNA copy number in Tfamflox/flox and Tfam cKO osteoclasts. mtDNA copy number per nuclear genome in osteoclasts was quantitated as described under “Experimental Procedures.” Genotypes are indicated. D, intracellular ATP levels in Tfamflox/flox and Tfam cKO osteoclasts. Intracellular ATP levels in Tfam cKO osteoclasts were significantly decreased when compared with Tfamflox/flox osteoclasts. E, time course of the survival of Tfamflox/flox and Tfam cKO osteoclasts. The number of TRAP-positive viable cells remaining at the different time points is shown as a percentage of the cells at time 0. The survival rate of Tfam cKO osteoclasts was significantly lower than Tfamflox/flox osteoclasts. F, Western blotting of cleaved caspase-3 using β-actin as an internal control. Tfam cKO osteoclasts exhibited the increased amount of cleaved caspase-3. G, bone-resorbing activity of Tfamflox/flox and Tfam cKO osteoclasts. After transfer onto dentine slices, osteoclasts were further incubated for 12 h. The resorption pits were visualized by staining with 1% toluidine blue, and the resorbed area was quantified using an image analysis system. The pit-forming activity of Tfam cKO osteoclasts was significantly higher than that of Tfamflox/flox osteoclasts. Representative resorption pits, visualized by toluidine blue staining, are also shown. Scale bar: 500 μm. *, p < 0.05, **, p < 0.01 versus normal Tfamflox/flox osteoclasts.

FIGURE 4.

AMPK activity does not affect osteoclast function.A, effect of intracellular ATP level on AMPK activity. Basal activity of AMPK was up-regulated in ATP-depleted osteoclasts after differentiation (left panel) or Tfam ablation (middle panel), whereas ATP-replete osteoclasts expressing Bcl-xL exhibited lower AMPK phosphorylation (right panel). AxGFP, adenovirus-mediated expression of GFP; AxBcl-xL, adenovirus-mediated expression of Bcl-xL. B, schematic representation of AMPKα1DN, AMPKα2DN, AMPKα1CA, and AMPKα2CA. C, adenovirus-mediated expression of AMPK mutants (AxAMPK) and modulation of AMPK activity in osteoclasts. The phosphorylation level of acetyl-CoA carboxylase (ACC) at Ser-79, a direct target of AMPK, was reduced by adenovirus-mediated expression of AMPKα1DN or AMPKα2DN, whereas induction of AMPKα1CA or AMPKα2CA dramatically increased acetyl-CoA carboxylase phosphorylation. D, time course of survival of osteoclasts expressing AMPK mutants. The number of TRAP-positive viable cells remaining at the different time points is shown as a percentage of the cells at time 0. Modulation of AMPK activity did not appear to affect osteoclast survival. E, bone-resorbing activity of osteoclasts expressing AMPK mutants. After transfer onto dentine slices, osteoclasts were further incubated for 12 h. No differences in the bone-resorbing activity were observed among osteoclasts expressing AMPK mutants.

FIGURE 5.

Extracellular ATP inhibits osteoclast function.A, the effects of intracellular ATP levels on constitutive ATP release. ATP release from ATP-depleted Tfam cKO osteoclasts was reduced (left panel), whereas ATP-replete osteoclasts expressing Bcl-xL showed enhanced basal ATP release (right panel). **, p < 0.01. AxGFP, adenovirus-mediated expression of GFP; AxBcl-xL, adenovirus-mediated expression of Bcl-xL. B, the effects of ATPγS (left panel) and BzATP (right panel) on the survival and bone-resorbing activity of osteoclasts. The addition of ATPγS and BzATP dramatically reduced osteoclast survival and bone resorption in a dose-dependent manner. *, p < 0.05, **, p < 0.01 versus untreated osteoclasts. C, morphological changes in osteoclasts stimulated with 100 μm ATPγS for 3 h (middle panel) or 150 μm BzATP for 30 min (bottom panel). The treatment with these ATP analogues caused morphological changes with numerous cytoplasmic vacuoles. Scale bar: 100 μm.

FIGURE 6.

Adenovirus-mediated Bcl-xL (AxBcl-xL) infection reversed the inhibitory effect of extracellular ATP on osteoclast survival but not on bone-resorbing activity.A, time course of the survival of Bcl-xL-expressing osteoclasts treated with 100 μm ATPγS or 150 μm BzATP. Bcl-xL expression completely reversed the inhibitory effect of ATPγS on osteoclast survival, whereas the survival curve of Bcl-xL-expressing osteoclasts showed a 30% reduction during the first 6 h after BzATP addition, after which stabilization occurred. **, p < 0.01 versus adenovirus-mediated expression of GFP (AxGFP)-infected osteoclasts. B, bone-resorbing activity of Bcl-xL-expressing osteoclasts treated with 100 μm ATPγS or 150 μm BzATP. Bone-resorbing activity of osteoclasts expressing Bcl-xL was significantly inhibited by treatments with ATPγS or BzATP, despite the improvement of survival rate. Representative resorption pits, visualized by toluidine blue staining, are also shown. **, p < 0.01.

FIGURE 7.

Extracellular ATP alters Pyk2 distribution in osteoclasts.A, Western blot analysis of fodrin, Pyk2, phospho-tyrosine, c-Src, and actin levels in osteoclasts treated with ATPγS or BzATP at different time points. Degradation in cytoskeletal proteins including α-fodrin, actin, Pyk2, and c-Src was not detected, and tyrosine-phosphorylated protein patterns remained unchanged upon ATPγS (100 μm) or BzATP (150 μm) stimulation. B, double immunofluorescence staining of F-actin (red) and Pyk2 (green) in osteoclasts after stimulation of ATPγS (100 μm) or BzATP (150 μm) for 1 h. Stimulation with ATPγS or BzATP disrupted the actin ring formation of osteoclasts and facilitated translocation of Pyk2 from the periphery to the cell center. Scale bar: 50 μm. C, Western blots of Triton X-100-insoluble cytoskeletons of osteoclasts treated with ATPγS (100 μm) or BzATP (150 μm) for 1 h. Note that the amount of Pyk2 stayed associated with the cytoskeletons was reduced after ATPγS or BzATP stimulation.

FIGURE 8.

Effects of removal of extracellular ATP or P2X7 receptor blockade on osteoclastic bone resorption.A, the effects of apyrase, which hydrolyzes extracellular ATP, on the survival and bone-resorbing activity of osteoclasts. Apyrase treatment up-regulated osteoclastic bone resorption with a slight increase in osteoclast survival. *, p < 0.05, **, p < 0.01 versus untreated osteoclasts. B, the effects of P2rx7 deficiency on the survival and bone-resorbing activity of osteoclasts. P2rx7 deficiency increased osteoclastic bone resorption with a tendency toward increased survival. *, p < 0.05, **, p < 0.01 versus normal P2rx7+/+ osteoclasts. C, morphological changes in Bcl-xL-expressing osteoclasts after a 48-h incubation. Note that Bcl-xL-induced morphological changes including large vacuoles and membrane blebs were reduced by apyrase treatment (10 units/ml) or P2rx7 deficiency. AxBcl-xL, adenovirus-mediated expression of Bcl-xL. Scale bar: 100 μm. D, bone-resorbing activity of P2rx7+/+ osteoclasts expressing Bcl-xL treated with apyrase and P2rx7−/− osteoclasts expressing Bcl-xL. The inhibitory effect of Bcl-xL expression on osteoclastic bone resorption was partially reversed by apyrase (10 units/ml) or P2rx7 deficiency. Representative resorption pits, visualized by toluidine blue staining, are also shown. AxGFP, adenovirus-mediated expression of GFP. **, p < 0.01.

FIGURE 9.

Age-related changes in osteoclasts and a model for the role of intracellular ATP levels in osteoclastic bone resorption.A and B, mtDNA copy number (A) and intracellular ATP levels (B) in osteoclasts derived from 3- and 24-month-old mice. mtDNA copy number and intracellular ATP levels were significantly decreased in the osteoclasts of 24-month-old mice. C and D, time course of the survival (C) and bone-resorbing activity of osteoclasts (D) derived from 3- and 24-month-old mice. The osteoclasts of 24-month-old mice exhibited increased bone resorption with a tendency toward shorter survival. Representative resorption pits, visualized by toluidine blue staining, are also shown. E, graphic showing how osteoclasts exhibit the inverse correlation between their survival and bone-resorbing activity. In osteoclasts, intracellular ATP levels is influenced by developmental, physiological, and pathological cues. Intracellular ATP content is one of the key players in regulating osteoclast survival. On the other hand, released ATP from intracellular stores has an inhibitory effect on cytoskeletal organization and osteoclastic bone resorption via purinergic receptors. The delicate functional balance of intracellular and extracellular ATP levels regulates osteoclast function. *, p < 0.05, **, p < 0.01 versus osteoclasts derived from 3-month-old mice.