Metabolic properties of the osteoclast

Osteoclasts are defined as cells capable of excavating 3-dimensional resorption pits in bone and other mineralised tissues. They are derived from the differentiation/fusion of promonocytic precursors, and are usually large, multinucleated cells. In common with other cells from this myeloid lineage such as macrophages and dendritic cells, they are adapted to function in hypoxic, acidic environments. The process of bone resorption is rapid and is presumably highly energy-intensive, since osteoclasts must actively extrude protons to dissolve hydroxyapatite mineral, whilst secreting cathepsin K to degrade collagen, as well as maintaining a high degree of motility. Osteoclasts are well known to contain abundant mitochondria but they are also able to rely on glycolytic (anaerobic) metabolism to generate the ATP needed to power their activity. Their primary extracellular energy source appears to be glucose. Excessive accumulation of mitochondrial reactive oxygen species in osteoclasts during extended periods of high activity in oxygen-poor environments may promote apoptosis and help to limit bone resorption — a trajectory that could be termed “live fast, die young”. In general, however, the metabolism of osteoclasts remains a poorly-investigated area, not least because of the technical challenges of studying actively resorbing cells in appropriate conditions

Activation of the P2Y2 receptor regulates bone cell function by enhancing ATP release

Bone cells constitutively release ATP into the extracellular environment where it acts locally via P2 receptors to regulate bone cell function. Whilst P2Y2 receptor stimulation regulates bone mineralisation, the functional effects of this receptor in osteoclasts remain unknown. This investigation used the P2Y2 receptor knockout (P2Y2R−/−) mouse model to investigate the role of this receptor in bone. MicroCT analysis of P2Y2R−/− mice demonstrated age-related increases in trabecular bone volume (≤48%), number (≤30%) and thickness (≤17%). In vitro P2Y2R−/− osteoblasts displayed a 3-fold increase in bone formation and alkaline phosphatase activity, whilst P2Y2R−/− osteoclasts exhibited a 65% reduction in resorptive activity. Serum cross-linked C-telopeptide levels (CTX, resorption marker) were also decreased (≤35%). The resorption defect in P2Y2R−/− osteoclasts was rescued by the addition of exogenous ATP, suggesting that an ATP deficit could be a key factor in the reduced function of these cells. In agreement, we found that basal ATP release was reduced up to 53% in P2Y2R−/− osteoclasts. The P2Y2 receptor agonists, UTP and 2-thioUTP, increased osteoclast activity and ATP release in wild-type but not in P2Y2R−/− cells. This indicates that the P2Y2 receptor may regulate osteoclast function indirectly by promoting ATP release. UTP and 2-thioUTP also stimulate ATP release from osteoblasts suggesting that the P2Y2 receptor exerts a similar function in these cells. Taken together, our findings are consistent with the notion that the primary action of P2Y2 receptor signalling in bone is to regulate extracellular ATP levels

Extracellular pyrophosphate: The body's “water softener”

Extracellular pyrophosphate (ePPi) was first identified as a key endogenous inhibitor of mineralisation in the 1960's by Fleisch and colleagues. The main source of ePPi seems to be extracellular ATP which is continually released from cells in a controlled way. ATP is rapidly broken down by enzymes including ecto-nucleotide pyrophosphatase/phosphodiesterases to produce ePPi. The major function of ePPi is to directly inhibit hydroxyapatite formation and growth meaning that this simple molecule acts as the body's own “water softener”. However, studies have also shown that ePPi can influence gene expression and regulate its own production and breakdown. This review will summarise our current knowledge of ePPi metabolism and how it acts to prevent pathological soft tissue calcification and regulate physiological bone mineralisation

Acidosis Is a Key Regulator of Osteoblast Ecto-Nucleotidase Pyrophosphatase/Phosphodiesterase 1 (NPP1) Expression and Activity

Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (Pi) to pyrophosphate (PPi) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both Pi and PPi, a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto‐nucleotidases. This study investigated the expression and activity of ecto‐nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto‐nucleotidases including NTPdase 1–6 (ecto‐nucleoside triphosphate diphosphohydrolase) and NPP1‐3 (ecto‐nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > alkaline phosphatase > ecto‐5‐nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8‐fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto‐nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (P < 0.001). Release of ATP, one of the key substrates for NPP1, from osteoblasts, was unaffected by acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5‐fold, P < 0.001) and was partially resistant to the inhibitory effect of acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to acid conditions

Pyrophosphate: a key inhibitor of mineralisation

Inorganic pyrophosphate has long been known as a by-product of many intracellular biosynthetic reactions, and was first identified as a key endogenous inhibitor of biomineralisation in the 1960s. The major source of pyrophosphate appears to be extracellular ATP, which is released from cells in a controlled manner. Once released, ATP can be rapidly hydrolysed by ecto-nucleotide pyrophosphatase/phosphodiesterases to produce pyrophosphate. The main action of pyrophosphate is to directly inhibit hydroxyapatite formation thereby acting as a physiological 'water-softener'. Evidence suggests pyrophosphate may also act as a signalling molecule to influence gene expression and regulate its own production and breakdown. This review will summarise our current understanding of pyrophosphate metabolism and how it regulates bone mineralisation and prevents harmful soft tissue calcification

Strontium potently inhibits mineralisation in bone-forming primary rat osteoblast cultures and reduces numbers of osteoclasts in mouse marrow cultures

The basic mechanisms by which strontium ranelate acts on bone are still unclear. We show that an important action of strontium salts is to block calcification in cultures of osteoblasts, the bone-forming cells. These results suggest that strontium treatment could have previously overlooked effects on bone

Extracellular Nucleotides Regulate Arterial Calcification by Activating Both Independent and Dependent Purinergic Receptor Signaling Pathways

Arterial calcification, the deposition of calcium-phosphate crystals in the extracellular matrix, resembles physiological bone mineralization. It is well-known that extracellular nucleotides regulate bone homeostasis raising an emerging interest in the role of these molecules on arterial calcification. The purinergic independent pathway involves the enzymes ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), 5′-nucleotidase and alkaline phosphatase. These regulate the production and breakdown of the calcification inhibitor—pyrophosphate and the calcification stimulator—inorganic phosphate, from extracellular nucleotides. Maintaining ecto-nucleotidase activities in a well-defined range is indispensable as enzymatic hyper- and hypo-expression has been linked to arterial calcification. The purinergic signaling dependent pathway focusses on the activation of purinergic receptors (P1, P2X and P2Y) by extracellular nucleotides. These receptors influence arterial calcification by interfering with the key molecular mechanisms underlying this pathology, including the osteogenic switch and apoptosis of vascular cells and possibly, by favoring the phenotypic switch of vascular cells towards an adipogenic phenotype, a recent, novel hypothesis explaining the systemic prevention of arterial calcification. Selective compounds influencing the activity of ecto-nucleotidases and purinergic receptors, have recently been developed to treat arterial calcification. However, adverse side-effects on bone mineralization are possible as these compounds reasonably could interfere with physiological bone mineralization

Chronic administration of Glucagon-like peptide-1 receptor agonists improves trabecular bone mass and architecture in ovariectomised mice

Some anti-diabetic therapies can have adverse effects on bone health and increase fracture risk. In this study, we tested the skeletal effects of chronic administration of two Glucagon-like peptide-1 receptor agonists (GLP-1RA), increasingly used for type 2 diabetes treatment, in a model of osteoporosis associated bone loss and examined the expression and activation of GLP-1R in bone cells. Mice were ovariectomised (OVX) to induce bone loss and four weeks later they were treated with Liraglutide (LIR) 0.3 mg/kg/day, Exenatide (Ex-4) 10 μg/kg/day or saline for four weeks. Mice were injected with calcein and alizarin red prior to euthanasia, to label bone-mineralising surfaces. Tibial micro-architecture was determined by micro-CT and bone formation and resorption parameters measured by histomorphometric analysis. Serum was collected to measure calcitonin and sclerostin levels, inhibitors of bone resorption and formation, respectively. GLP-1R mRNA and protein expression were evaluated in the bone, bone marrow and bone cells using RT-PCR and immunohistochemistry. Primary osteoclasts and osteoblasts were cultured to evaluate the effect of GLP-1RA on bone resorption and formation in vitro. GLP-1RA significantly increased trabecular bone mass, connectivity and structure parameters but had no effect on cortical bone. There was no effect of GLP-1RA on bone formation in vivo but an increase in osteoclast number and osteoclast surfaces was observed with Ex-4. GLP-1R was expressed in bone marrow cells, primary osteoclasts and osteoblasts and in late osteocytic cell line. Both Ex-4 and LIR stimulated osteoclastic differentiation in vitro but slightly reduced the area resorbed per osteoclast. They had no effect on bone nodule formation in vitro. Serum calcitonin levels were increased and sclerostin levels decreased by Ex-4 but not by LIR. Thus, GLP-1RA can have beneficial effects on bone and the expression of GLP-1R in bone cells may imply that these effects are exerted directly on the tissue

Role of the P2Y(13) Receptor in the Differentiation of Bone Marrow Stromal Cells into Osteoblasts and Adipocytes

Accumulating evidence indicates that extracellular nucleotides, signaling through purinergic receptors, play a significant role in bone remodeling. Mesenchymal stem cells (MSCs) express functional P2Y receptors whose expression level is regulated during osteoblast or adipocyte differentiation. P2Y13-deficient mice were previously shown to exhibit a decreased bone turnover associated with a reduction in the number of both osteoblasts and osteoclasts on the bone surfaces. We therefore examined whether P2Y13R activation was involved in the osteogenic differentiation of MSC. Our study demonstrated that ADP stimulation of P2Y13R+/+ (but not P2Y13R-/-) adherent bone marrow stromal cells (BMSCs) increased significantly the formation of alkaline phosphatase-colony-forming units (CFU-ALP) as well as the expression of osteoblastic markers (osterix, alkaline phosphatase, and collagen I) involved in the maturation of preosteoblasts into osteoblasts. The number of CFU-ALP obtained from P2Y13R-/- BMSC and the level of osteoblastic gene expression after osteogenic stimulation were strongly reduced compared to those obtained in wild-type cell cultures. In contrast, when P2Y13R-/- BMSCs were incubated in an adipogenic medium, the number of adipocytes generated and the level of adipogenic gene expression (PPARγ2 and Adipsin) were higher than those obtained in P2Y13R+/+ MSC. Interestingly, we observed a significant increase of the number of bone marrow adipocytes in tibia of P2Y13R-/- mice. In conclusion, our findings indicate that the P2Y13R plays an important role in the balance of osteoblast and adipocyte terminal differentiation of bone marrow progenitors. Therefore, the P2Y13 receptor can be considered as a new pharmacological target for the treatment of bone diseases like osteoporosis

Lack of effect of adenosine on the function of rodent osteoblasts and osteoclasts in vitro

Extracellular ATP, signalling through P2 receptors, exerts well-documented effects on bone cells, inhibiting mineral deposition by osteoblasts and stimulating the formation and resorptive activity of osteoclasts. The aims of this study were to determine the potential osteotropic effects of adenosine, the hydrolysis product of ATP, on primary bone cells in vitro. We determined the effect of exogenous adenosine on (1) the growth, alkaline phosphatase (TNAP) activity and bone-forming ability of osteoblasts derived from the calvariae of neonatal rats and mice and the marrow of juvenile rats and (2) the formation and resorptive activity of osteoclasts from juvenile mouse marrow. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed marked differences in the expression of P1 receptors in osteoblasts from different sources. Whilst mRNA for the A1 and A2B receptors was expressed by all primary osteoblasts, A2A receptor expression was limited to rat bone marrow and mouse calvarial osteoblasts and the A3 receptor to rat bone marrow osteoblasts. We found that adenosine had no detectable effects on cell growth, TNAP activity or bone formation by rodent osteoblasts in vitro. The analogue 2-chloroadenosine, which is hydrolysed more slowly than adenosine, had no effects on rat or mouse calvarial osteoblasts but increased TNAP activity and bone formation by rat bone marrow osteoblasts by 30–50 % at a concentration of 1 μM. Osteoclasts were found to express the A2A, A2B and A3 receptors; however, neither adenosine (≤100 μM) nor 2-chloroadenosine (≤10 μM) had any effect on the formation or resorptive activity of mouse osteoclasts in vitro. These results suggest that adenosine, unlike ATP, is not a major signalling molecule in the bone