Dissolution of the inorganic phase of bone leading to release of calcium regulates osteoclast survival

doi:10.1016/j.bbrc.2007.06.145

Biochemical and Biophysical Research Communications

Volume 360, Issue 4, 7 September 2007, Pages 834-839

https://doi.org/10.1016/j.bbrc.2007.06.145 Get rights and content

Abstract

Osteoclasts are the sole cells possessing the ability to resorb calcified bone matrix. This occurs via secretion of hydrochloric acid mediated by the V-ATPase and the chloride channel ClC-7. Loss of acidification leads to osteopetrosis characterized by ablation of bone resorption and increased osteoclast numbers, indicating increased life span of the osteoclasts. To investigate the role of the inorganic phase of bone with respect to osteoclast life span, we used the V-ATPase inhibitor bafilomycin and the calcium uptake antagonist ryanodine on human osteoclasts cultured on calcified and decalcified bone slices. Bafilomycin inhibited bone resorption and increased osteoclast survival on calcified but not decalcified bones. Ryanodine attenuated calcium uptake and thereby augmented osteoclast survival on calcified bones. In summary, we found that acidification leading to calcium release from bone during resorption controls osteoclast survival, potentially explaining the increased numbers of osteoclasts in patients with osteopetrosis.

Section snippets

Materials and methods

Drugs and chemicals. Bafilomycin A1 was purchased from Calbiochem. Ryanodine was purchased from Biomol. The culture medium was from Life Technologies. The remaining chemicals were from Sigma–Aldrich, unless otherwise specified.

Cell culture. The CD14+ isolation was performed as previously described [18]. Briefly, the monocytes were isolated from peripheral blood by centrifugation on a Ficoll-Paque gradient (Amersham Pharmacia) and magnetically sorted using a CD14+ magnetic bead isolation kit

Inhibition of acidification promotes osteoclast survival on calcified bones, but not decalcified bones

Patients with defective acidification of the resorption lacuna have increased numbers of non-resorbing osteoclasts [20]. We investigated the effect of inhibition of the V-ATPase with respect to survival of osteoclasts seeded on calcified and decalcified bone slices. We found that bafilomycin at 20 nM inhibited bone resorption of calcified bone completely (Fig. 1A) and that it augmented osteoclast survival compared to non-treated osteoclasts by 250–300% (p < 0.001) on day 7 and 11, examined by

Discussion

By using a novel approach focusing on the role of the inorganic phase of bone on osteoclast survival, we described that osteoclast life span is regulated by dissolution of the inorganic matrix leading to Ca²⁺ release, which subsequently induces apoptosis of the osteoclasts, and thereby controls the resorptive activity of the cells. These results explain the increased numbers of osteoclast seen in osteopetrotic patients with defective acidification of the resorption lacunae [20], [22].

References (25)

U. Kornak et al.
Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man
Cell
(2001)
K. Henriksen et al.
Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II
Am. J. Pathol.
(2004)
M.A. Karsdal et al.
Acidification of the osteoclastic resorption compartment provides insight into the coupling of bone formation to bone resorption
Am. J. Pathol.
(2005)
I.A. Silver et al.
Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts
Exp. Cell Res.
(1988)
F. Lorget et al.
High extracellular calcium concentrations directly stimulate osteoclast apoptosis
Biochem. Biophys. Res. Commun.
(2000)
M.A. Karsdal et al.
TGF-beta controls human osteoclastogenesis through the p38 MAP kinase and regulation of RANK expression
J. Biol. Chem.
(2003)
J. Bollerslev et al.
Ultrastructural investigations of bone resorptive cells in two types of autosomal dominant osteopetrosis
Bone
(1993)
M. Zaidi et al.
‘Calcium-activated’ intracellular calcium elevation: a novel mechanism of osteoclast regulation
Biochem. Biophys. Res. Commun.
(1989)
H.K. Vaananen et al.
The cell biology of osteoclast function
J. Cell Sci.
(2000)
R. Baron et al.
Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kDa lysosomal membrane protein at the osteoclast ruffled border
J. Cell Biol.
(1985)

Y.P. Li et al.

Atp6i-deficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification

Nat. Genet.

(1999)

A. Frattini et al.

Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis

Nat. Genet.

(2000)

Cited by (25)

Sustained local ionic homeostatic imbalance caused by calcification modulates inflammation to trigger heterotopic ossification
2022, Acta Biomaterialia
Citation Excerpt :
Sensing is made mostly via CaSR [264–268], but other receptors have been identified, such as the notch receptor and cadherins [269,270]. A change of [Ca2+]e affects osteoclasts intracellular calcium levels [266,271,272], attachment [266,272,273], resorptive activity [265–268], apoptosis [233,274], differentiation [233], and genesis [265]. Accordingly, Hwang and Putney [275] suggested that [Ca2+]e regulates the resorptive activities of osteoclasts.
Heterotopic ossification (HO) is a condition triggered by an injury leading to the formation of mature lamellar bone in extraskeletal soft tissues. Despite being a frequent complication of orthopedic and trauma surgery, brain and spinal injury, the etiology of HO is poorly understood. The aim of this study is to evaluate the hypothesis that a sustained local ionic homeostatic imbalance (SLIHI) created by mineral formation during tissue calcification modulates inflammation to trigger HO. This evaluation also considers the role SLIHI could play for the design of cell-free, drug-free osteoinductive bone graft substitutes. The evaluation contains five main sections. The first section defines relevant concepts in the context of HO and provides a summary of proposed causes of HO. The second section starts with a detailed analysis of the occurrence and involvement of calcification in HO. It is followed by an explanation of the causes of calcification and its consequences. This allows to speculate on the potential chemical modulators of inflammation and triggers of HO. The end of this second section is devoted to in vitro mineralization tests used to predict the ectopic potential of materials. The third section reviews the biological cascade of events occurring during pathological and material-induced HO, and attempts to propose a quantitative timeline of HO formation. The fourth section looks at potential ways to control HO formation, either acting on SLIHI or on inflammation. Chemical, physical, and drug-based approaches are considered. Finally, the evaluation finishes with a critical assessment of the definition of osteoinduction.
The ability to regenerate bone in a spatially controlled and reproducible manner is an essential prerequisite for the treatment of large bone defects. As such, understanding the mechanism leading to heterotopic ossification (HO), a condition triggered by an injury leading to the formation of mature lamellar bone in extraskeletal soft tissues, would be very useful. Unfortunately, the mechanism(s) behind HO is(are) poorly understood. The present study reviews the literature on HO and based on it, proposes that HO can be caused by a combination of inflammation and calcification. This mechanism helps to better understand current strategies to prevent and treat HO. It also shows new opportunities to improve the treatment of bone defects in orthopedic and dental procedures.
Long-term therapy with intravenous zoledronate increases the number of nonattached osteoclasts
2017, Journal of Cranio-Maxillofacial Surgery
Citation Excerpt :
According to Yang et al. (2005) increased osteoclast numbers could result if BP-treated osteoclasts, although poorly functional, persist longer than normal osteoclasts. The released calcium is an important signal for osteoclast death (Nielsen et al., 2007). According to Weinsten et al. (2009) inhibition of bone resorption by nitrogen-containing BPs would effectively diminish the signal for osteoclast death that results from calcium release during resorption, thereby prolonging their lifespan and allowing time for fusion with additional mononuclear progenitors.
The purpose of this study was to investigate the influence of long-term therapy with intravenous zoledronate (ZA) on the healing of extraction sockets in rats. Forty rats, divided into groups C (Control) and Z (Zoledronate), received intravenous injections of either saline solution or ZA for 24 weeks. Their right maxillary incisor was extracted. Euthanasia was performed at 7 or 28 days postoperative. Histomorphometric (Newly Formed Bone Area) and immunohistochemical (RANKL, OPG and TRAP) analyses were performed. Data were statistically analyzed (ANOVA, Tukey's test and Kruskal–Wallis, Dunn's Multiple Comparison test).Groups C and Z showed similar new bone area, RANKL and OPG immunolabeling. The number of TRAP-positive multinucleated cells was significantly higher in Group Z than in Group C at 28 days. A significantly higher proportion of nonattached osteoclasts were seen in Group Z than in Group C at both periods of analysis. Long-term therapy with intravenous ZA stimulated nonattached osteoclast formation in extraction sockets in rats, thus decreasing local bone resorption. However, it did not influence bone formation by osteoblasts.
Lentiviral gene transfer of TCIRG1 into peripheral blood CD34<sup>+</sup> cells restores osteoclast function in infantile malignant osteopetrosis
2013, Bone
Citation Excerpt :
This clearly establishes that the introduction of non-mutated TCIRG1 into IMO osteoclasts restores their ability to acidify the resorption lacunae and resorb bone in vitro. Furthermore, in the IMO cells the TRACP levels are increased, a phenomenon which previously has been shown to be related to increased survival of the osteoclasts due to their lowered resorption activity [21,25,26]. Importantly, this process is not specific to lack of TCIRG1 as it is also observed in c-src, cathepsin K and ClC-7 deficient osteoclasts [4,27–30].
Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by non-functional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to restore the resorptive function of IMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1 cDNA. CD34⁺ cells from peripheral blood of five IMO patients and from normal cord blood were transduced with lentiviral vectors expressing TCIRG1 and GFP under a SFFV promoter, expanded in culture and differentiated on bone slices to mature osteoclasts. qPCR analysis and western blot revealed increased mRNA and protein levels of TCIRG1, comparable to controls. Vector corrected IMO osteoclasts generated increased release of Ca^2 + and bone degradation product CTX-I into the media as well as increased formation of resorption pits in the bone slices, while non-corrected IMO osteoclasts failed to resorb bone. Resorption was approximately 70–80% of that of osteoclasts generated from cord blood. Furthermore, transduced CD34⁺ cells successfully engrafted in NSG-mice. In conclusion we provide the first evidence of lentiviral-mediated correction of a human genetic disease affecting the osteoclastic lineage.
A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts
2012, Bone
Citation Excerpt :
An interesting aspect in relation to the data from osteoclasts on different matrices is whether calcium in itself is part of the anabolic signal from osteoclasts to osteoblasts. Calcium is known to reduce osteoclast survival [55], and could also be a part of induction of new bone formation [57], although a direct link between calcium released during resorption and bone formation remains to be proven. On the other hand we also observed that diphyllin and E64 reduced the anabolic potential of osteoclasts by similar amounts, which could indicate that calcium is not a major player, as E64 reduces calcium release only by 40% [47].
Osteoclasts are known to be important for the coupling process between bone resorption and formation.
The aim of this study was to address when osteoclasts are anabolically active.
Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium was collected from macrophages, pre-osteoclasts, and mature functional or non-resorbing osteopetrotic osteoclasts on either bone, plastic, decalcified bone or dentine with or without diphyllin, E64 or GM6001. Osteoclasts numbers were measured by TRACP activity. Bone resorption was evaluated by CTX-I and calcium release. The osteoblastic cell line 2 T3 was treated with 50% of CM or non-CM for 12 days. Bone formation was assessed by Alizarin Red extraction.
CM from mature osteoclasts induced bone formation, while CM from macrophages did not. Non-resorbing osteoclasts generated from osteopetrosis patients showed little resorption, but still an induction of bone formation by osteoblasts. Mimicking the reduction in bone resorption using the V-ATPase inhibitor Diphyllin, the cysteine proteinase inhibitor E64 and the MMP-inhibitor GM6001 showed that CM from diphyllin and E64 treated osteoclasts showed reduced ability to induce bone formation compared to CM from vehicle treated osteoclasts, while CM from GM6001 treated osteoclasts equaled vehicle CM. Osteoclasts on either dentine or decalcified bone showed strongly attenuated anabolic capacities.
In conclusion, we present evidence that osteoclasts, both dependent and independent of their resorptive activity, secrete factors stimulating osteoblastic bone formation.
Severe developmental bone phenotype in ClC-7 deficient mice
2010, Developmental Biology
Bone development is dependent on the functionality of three essential cell types: chondrocytes, osteoclasts and osteoblasts. If any of these cell types is dysfunctional, a developmental bone phenotype can result.
The bone disease osteopetrosis is caused by osteoclast dysfunction or impaired osteoclastogenesis, leading to increased bone mass. In ClC-7 deficient mice, which display severe osteopetrosis, the osteoclast malfunction is due to abrogated acidification of the resorption lacuna. This study sought to investigate the consequences of osteoclast malfunction on bone development, bone structure and bone modeling/remodeling in ClC-7 deficient mice. Bones from wildtype, heterozygous and ClC-7 deficient mice were examined by bone histomorphometry and immunohistochemistry.
ClC-7 deficient mice were found to have a severe developmental bone phenotype, characterized by dramatically increased bone mass, a high content of cartilage remnants, impaired longitudinal and radial growth, as well as lack of compact cortical bone development. Indices of bone formation were reduced in ClC-7 deficient mice; however, calcein labeling indicated that mineralization occurred on most trabecular bone surfaces. Osteoid deposition had great regional variance, but an osteopetrorickets phenotype, as observed in oc/oc mice, was not apparent in the ClC-7 deficient mice. A striking finding was the presence of very large abnormal osteoclasts, which filled the bone marrow space within the ClC-7 deficient bones. The development of these giant osteoclasts could be due to altered cell fate of the ClC-7 deficient osteoclasts, caused by increased cellular fusion and/or prolonged osteoclast survival.
In summary, malfunctional ClC-7 deficient osteoclasts led to a severe developmental bone phenotype including abnormally large and non-functional osteoclasts. Bone formation paremeters were reduced; however, bone formation and mineralization were found to be heterogenous and continuing.
Local communication on and within bone controls bone remodeling
2009, Bone
Bone remodeling is required for healthy calcium homeostasis and for repair of damage occurring with stress and age. Osteoclasts resorb bone and osteoblasts form bone. These processes normally occur in a tightly regulated sequence of events, where the amount of formed bone equals the amount of resorbed bone, thereby restoring the removed bone completely. Osteocytes are the third cell type playing an essential role in bone turnover. They appear to regulate activation of bone remodeling, and they exert both positive and negative regulation on both osteoclasts and osteoblasts.
In this review, we consider the intricate communication between these bone cells in relation to bone remodeling, reviewing novel data from patients with mutations rendering different cell populations inactive, which have shown that these interactions are more complex than originally thought. We highlight the high probability that a detailed understanding of these processes will aid in the development of novel treatments for bone metabolic disorders, i.e. we discuss the possibility that bone resorption can be attenuated pharmacologically without a secondary reduction in bone formation.

View all citing articles on Scopus

View full text

Dissolution of the inorganic phase of bone leading to release of calcium regulates osteoclast survival

Abstract

Section snippets

Materials and methods

Inhibition of acidification promotes osteoclast survival on calcified bones, but not decalcified bones

Discussion

Cell

Am. J. Pathol.

Am. J. Pathol.

Exp. Cell Res.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Bone

Biochem. Biophys. Res. Commun.

The cell biology of osteoclast function

J. Cell Sci.

Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kDa lysosomal membrane protein at the osteoclast ruffled border

J. Cell Biol.

Atp6i-deficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification

Nat. Genet.

Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis

Nat. Genet.