ATP and UTP at low concentrations strongly inhibit bone formation by osteoblasts: a novel role for the P2Y2 receptor in bone remodeling

There is increasing evidence that extracellular nucleotides act on bone cells via multiple P2 receptors. The naturally-occurring ligand ATP is a potent agonist at all receptor subtypes, whereas ADP and UTP only act at specific receptor subtypes. We have reported that the formation and resorptive activity of rodent osteoclasts are stimulated powerfully by both extracellular ATP and its first degradation product, ADP, the latter acting at nanomolar concentrations, probably via the P2Y1 receptor subtype. In the present study, we investigated the actions of ATP, ADP, adenosine, and UTP on osteoblastic function. In 16-21 day cultures of primary rat calvarial osteoblasts, ADP and the selective P2Y1 agonist 2-methylthioADP were without effect on bone nodule formation at concentrations between 1 and 125 microM, as was adenosine. However, UTP, a P2Y2 and P2Y4 receptor agonist, known to be without effect on osteoclast function, strongly inhibited bone nodule formation at concentrations >or= 1 microM. ATP was inhibitory at >or= 10 microM. Rat osteoblasts express P2Y2, but not P2Y4 receptor mRNA, as determined by in situ hybridization. Thus, the low-dose effects of extracellular nucleotides on bone formation and bone resorption appear to be mediated via different P2Y receptor subtypes: ADP, signalling through the P2Y1 receptor on both osteoclasts and osteoblasts, is a powerful stimulator of osteoclast formation and activity, whereas UTP, signalling via the P2Y2 receptor on osteoblasts, blocks bone formation by osteoblasts. ATP, the 'universal' agonist, can simultaneously stimulate resorption and inhibit bone formation. These findings suggest that extracellular nucleotides could function locally as important negative modulators of bone metabolism, perhaps contributing to bone loss in a number of pathological states.     

Extracellular ADP is a powerful osteolytic agent: evidence for signaling through the P2Y(1) receptor on bone cells.

There is increasing evidence that extracellular nucleotides act on bone cells via P2 receptors. This study investigated the action of ADP and 2-methylthioADP, a potent ADP analog with selectivity for the P2Y(1) receptor, on osteoclasts, the bone-resorbing multinuclear cells. Using three different assays, we show that ADP and 2-methylthioADP at nanomolar to submicromolar levels caused up to fourfold to sixfold increases in osteoclastic bone resorption. On mature rat osteoclasts, cultured for 1 day on polished dentine disks, peak effects on resorption pit formation were observed between 20 nM and 2 microM of ADP. The same concentrations of ADP also stimulated osteoclast and resorption pit formation in 10-day mouse marrow cultures on dentine disks. In 3-day explant cultures of mouse calvarial bones, the stimulatory effect of ADP on osteoclast-mediated Ca(2+) release was greatest at 5-50 microM and equivalent to the maximal effects of prostaglandin E(2). The ADP effects were blocked in a nontoxic manner by MRS 2179, a P2Y(1) receptor antagonist. Using in situ hybridization and immunocytochemistry, we found evidence for P2Y(1) receptor expression on both osteoclasts and osteoblasts; thus, ADP could exert its actions both directly on osteoclasts and indirectly via P2Y(1) receptors on osteoblasts. As a major ATP degradation product, ADP is a novel stimulator of bone resorption that could help mediate inflammatory bone loss in vivo.     

PPAR agonists stimulate adipogenesis at the expense of osteoblast differentiation while inhibiting osteoclast formation and activity

Drugs used in the treatment of type 2 diabetes and cardiovascular disease, specifically peroxisome proliferator‐activated receptor (PPAR) agonists, have been reported to affect bone cell function and fracture risk. In this study, we assessed the direct effects of PPAR‐γ agonists (rosiglitazone and troglitazone), used in the treatment of diabetes, and a PPAR‐α agonist (fenofibrate), used to treat hyperlipidaemia, on the function of primary osteoblasts and osteoclasts. Formation of ‘trabecular’ bone structures by rat calvarial osteoblasts was reduced by up to 85% in cultures treated with rosiglitazone and by 45% in troglitazone‐treated or fenofibrate‐treated cultures; at the same time, lipid droplet formation was increased by 40–70%. The expression of key osteogenic markers was similarly downregulated in cultures treated with PPAR agonists, whereas adipogenesis markers were upregulated. Formation of osteoclasts in cultures derived from mouse marrow diminished with fenofibrate treatment, whereas both glitazones reduced resorptive activity without affecting osteoclast number. Metformin, although not a PPAR agonist, is also commonly used in the treatment of type 2 diabetes. Here, metformin was found to have no effect on bone cell function. Taken together, these data suggest that PPAR‐γ agonists may enhance bone loss via increased adipogenesis at the expense of osteoblast formation. In contrast, PPAR‐α agonists may prevent bone loss. Given that the prevalence of diabetes and cardiovascular disease is expected to rise significantly, greater attention may need to be paid to the effects of PPAR agonists on bone homeostasis. Copyright © 2014 John Wiley & Sons, Ltd.     

Licochalcone A inhibits the formation and bone resorptive activity of osteoclasts

Licochalcone A on the formation and bone resorptive activity of osteoclasts up to 5muM significantly inhibited the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL)-induced activity of tartrate-resistant acid phosphatase (TRAP) activity and formation of osteoclasts without any effect on cell viability. Interestingly, licochalcone A was shown to inhibit the RANKL-induced activation of extracellular signal-regulated kinase, translocation of NF-kappaB into nucleus and mRNA expression of Fra-2. Licochalcone A also inhibited the bone resorptive activity of mature osteoclasts and the expression of bone resorption-related genes. Inhibitory effects of licochalcone A on the formation and bone resorptive activity of mouse bone marrow macrophage-derived osteoclasts were also observed. In conclusion, licochalcone A has the potential to inhibit the formation of osteoclasts as well as the bone resorptive activity of mature osteoclasts.     

Hypoxia is a major stimulator of osteoclast formation and bone resorption

Hypoxia is known to act as a general stimulator of cells derived from marrow precursors. We investigated the effect of oxygen tension on the formation and function of osteoclasts, the cells responsible for bore resorption, which are of promonocytic origin. Using 7- and 13-day cultures of mouse marrow cells on ivory discs, we found that reducing oxygen tension from the ambient atmospheric level of 20% by increasing the proportion of nitrogen caused progressive increases in the formation of multinucleated osteoclasts and resorption pits. Peak effects occurred in 2% oxygen, where stimulations of resorption up to 21-fold were measured. Significant stimulations of osteoclast formation and resorption were observed even in severely hypoxic cultures gassed with 0.2% oxygen. Short-term cultures of cells disaggregated from rat bones indicated that hypoxia did not alter the resorptive activity of mature osteoclasts, but reduced their survival or adherence. In 3-day organ cultures of mouse calvarial bones, exposure to 2% oxygen resulted in maximal, fivefold stimulation of osteoclast-mediated calcium release, an effect equivalent to that of prostaglandin E(2) (PGE(2)), a reference osteolytic agent. Hypoxia also caused a moderate acidosis in calvarial cultures, presumably as a result of increased anaerobic metabolism; this observation is significant because osteoclast activation is dependent on extracellular acidification. Our experiments reveal a previously-overlooked mechanism of considerable potential importance for the regulation of bone destruction. These findings may help explain the bone loss associated with a wide range of pathological states involving local or systemic hypoxia, and emphasize the importance of the vasculature in bone.     

Tissue-nonspecific alkaline phosphatase promotes axonal growth of hippocampal neurons

Axonal growth is essential for establishing neuronal circuits during brain development and for regenerative processes in the adult brain. Unfortunately, the extracellular signals controlling axonal growth are poorly understood. Here we report that a reduction in extracellular ATP levels by tissue-nonspecific alkaline phosphatase (TNAP) is essential for the development of neuritic processes by cultured hippocampal neurons. Selective blockade of TNAP activity with levamisole or specific TNAP knockdown with short hairpin RNA interference inhibited the growth and branching of principal axons, whereas addition of alkaline phosphatase (ALP) promoted axonal growth. Neither activation nor inhibition of adenosine receptors affected the axonal growth, excluding the contribution of extracellular adenosine as a potential hydrolysis product of extracellular ATP to the TNAP-mediated effects. TNAP was colocalized at axonal growth cones with ionotropic ATP receptors (P2X₇ receptor), whose activation inhibited axonal growth. Additional analyses suggested a close functional interrelation of TNAP and P2X₇ receptors whereby TNAP prevents P2X₇ receptor activation by hydrolyzing ATP in the immediate environment of the receptor. Furthermore inhibition of P2X₇ receptor reduced TNAP expression, whereas addition of ALP enhanced P2X₇ receptor expression. Our results demonstrate that TNAP, regulating both ligand availability and protein expression of P2X₇ receptor, is essential for axonal development.     

Hypoxia stimulates vesicular ATP release from rat osteoblasts

Many neuronal and non‐neuronal cell types release ATP in a controlled manner. After release, extracellular ATP (or, following hydrolysis, ADP) acts on cells in a paracrine manner via P2 receptors. Extracellular nucleotides are now thought to play an important role in the regulation of bone cell function. ATP (and ADP), acting via the P2Y₁ receptor, stimulate osteoclast formation and activity, whilst P2Y₂ receptor stimulation by ATP (or UTP) inhibits bone mineralization by osteoblasts. We found that rat calvarial osteoblasts released ATP constitutively, in a differentiation‐dependent manner, with mature, bone‐forming osteoblasts releasing up to sevenfold more ATP than undifferentiated, proliferating cells. The inhibitors of vesicular exocytosis, monensin, and N‐ethylmaleimide (1–1,000 µM) inhibited basal ATP release by up to 99%. The presence of granular ATP‐filled vesicles within the osteoblast cytoplasm was demonstrated by quinacrine staining. Exposure to hypoxia (2% O₂) for up to 3 min increased ATP release from osteoblasts up to 2.5‐fold without affecting cell viability. Peak concentrations of ATP released into culture medium were >1 µM, which equates with concentrations known to exert significant effects on osteoblast and osteoclast function. Monensin and N‐ethylmaleimide (100 µM) attenuated the hypoxia‐induced ATP release by up to 80%. Depletion of quinacrine‐stained vesicles was also apparent after hypoxic stimulation, indicating that ATP release had taken place. These data suggest that vesicular exocytosis is a key mediator of ATP release from osteoblasts, in biologically significant amounts. Moreover, increased extracellular ATP levels following acute exposure to low O₂ could influence local purinergic signaling and affect the balance between bone formation and bone resorption. J. Cell. Physiol. 220: 155–162, 2009. © 2009 Wiley‐Liss, Inc.     

Regulation of new osteoclast formation by a bone cell-derived macromolecular factor

Medium conditioned by incubation with embryonic chick calvarial bones, which contain osteoblasts but not osteoclasts, stimulated new osteoclast formation in foetal long bone cultures and in adult bone marrow cultures formation of tartrate-resistant acid phosphatase (TRAP) positive cells was greatly stimulated. We have termed the factor responsible for this activity osteoclast growth/inducing factor (OGF). OGF was soluble, heat-stable and of size greater than 10kda. OGF activity was present also in mouse bone conditioned medium and in extracts of demineralized cortical diaphyseal bone of five-week-old chickens. OGF appeared to differ from the osteoblast-derived bone-resorbing factors previously observed as well as from macrophage colony stimulating factor (CSF-1). It is therefore probable that different locally secreted factors independently regulate the formation of osteoclasts and their activity.     

Expression of estrogen receptor-alpha in cells of the osteoclastic lineage

 Estrogen deficiency at the menopause is associated with an increased rate of bone loss and subsequent risk of skeletal fracture. Whilst cells of the osteoblastic lineage are known to express estrogen receptors, the presence of estrogen receptors in osteoclasts remains controversial. We have examined expression of the classic estrogen receptor, estrogen receptor-alpha (ERα), during osteoclast differentiation. In situ mRNA hybridisation with a digoxygenin-labelled riboprobe to ERα mRNA, together with immunocytochemical analysis using a human ERα-specific monoclonal antibody demonstrated similar findings and confirmed the expression of ERα in chondroblasts and osteoblasts from human fetal bone and mineralising human bone marrow cultures. ERα expression was detected in human bone marrow cultures treated with 1,25(OH)₂D₃ and macrophage colony-stimulating factor and in macrophage cultures treated with 1,25(OH)₂D₃. However, in an in vitro model of human osteoclast formation, no ERα expression was observed in the osteoclasts that developed. The human preosteoclast TCG 51 cell line showed strong expression of ERα in contrast to the low levels observed in the more mature bone resorptive TCG 23 cell line. No expression was detectable in osteoclasts cultured from giant cell tumour of bone (GCTB) tissue or in osteoclasts in Pagetic, GCTB, or hyperparathyroid bone tissues. In conclusion, preosteoclasts express detectable levels of ERα, but osteoclast maturation and bone resorption is associated with loss of ERα expression. This indicates that ERα expression and regulation may play a role in osteoclast formation. Accepted: 4 November 1998     

Bone morphogenetic protein 2 enhances mouse osteoclast differentiation via increased levels of receptor activator of NF-κB ligand expression in osteoblasts

1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] induces osteoclast formation via induction of receptor activator of NF-κB ligand (RANKL, also called TNF-related activation-induced cytokine: TRANCE) in osteoblasts. In cocultures of mouse bone marrow cells and osteoblasts, 1,25(OH)₂D₃ induced osteoclast formation in a dose-dependent manner, with maximum osteoclast formation observed at concentrations greater than 10^?9 M of 1,25(OH)₂D₃. In the presence of bone morphogenetic protein 2 (BMP-2), the maximum formation of osteoclasts was seen with lower concentrations of 1,25(OH)₂D₃ (greater than 10^?11 M), suggesting that BMP-2 enhances osteoclast formation induced by 1,25(OH)₂D₃. In addition, the expressions of RANKL mRNA and proteins were induced by 1,25(OH)₂D₃ in osteoblasts, and further upregulated by BMP-2. In mouse bone marrow cell cultures without 1,25(OH)₂D₃, BMP-2 did not enhance osteoclast differentiation induced by recombinant RANKL and macrophage colony-stimulating factor (M-CSF), indicating that BMP-2 does not target osteoclast precursors. Furthermore, BMP-2 up-regulated the expression level of vitamin D receptor (VDR) in osteoblasts. These results suggest that BMP-2 regulates mouse osteoclast differentiation via upregulation of RANKL in osteoblasts induced by 1,25(OH)₂D₃.     

Osteoblast tissue-nonspecific alkaline phosphatase antagonizes and regulates PC-1

Tissue-nonspecific alkaline phosphatase (TNAP) is essential for bone matrix mineralization, but the central mechanism for TNAP action remains undefined. We observed that ATP-dependent (45)Ca precipitation was decreased in calvarial osteoblast matrix vesicle (MV) fractions from TNAP-/- mice, a model of infantile hypophosphatasia. Because TNAP hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP(i)), we assessed phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH) activity, which hydrolyzes ATP to generate PP(i). Plasma cell membrane glycoprotein-1 (PC-1), but not the isozyme B10 (also called PDNP3) colocalized with TNAP in osteoblast MV fractions and pericellular matrix. PC-1 but not B10 increased MV fraction PP(i) and inhibited (45)Ca precipitation by MVs. TNAP directly antagonized inhibition by PC-1 of MV-mediated (45)Ca precipitation. Furthermore, the PP(i) content of MV fractions was greater in cultured TNAP-/- than TNAP+/+ calvarial osteoblasts. Paradoxically, transfection with wild-type TNAP significantly increased osteoblast MV fraction NTPPPH. Specific activity of NTPPPH also was twofold greater in MV fractions of osteoblasts from TNAP+/+ mice relative to TNAP-/- mice. Thus TNAP attenuates PC-1/NTPPPH-induced PP(i) generation that would otherwise inhibit MV-mediated mineralization. TNAP also paradoxically regulates PC-1 expression and NTPPPH activity in osteoblasts.     

Hypothermia inhibits osteoblast differentiation and bone formation but stimulates osteoclastogenesis

It has long been known that core body temperature declines with age, with temperatures of 35.5°C or below common in the elderly. However, the effects of temperature reduction on bone cell function and skeletal homeostasis have been little studied. We investigated the effects of mild hypothermia (35.5°C) and severe hypothermia (34°C) on bone-forming osteoblasts, and bone-resorbing osteoclasts. Formation of 'trabecular' bone structures by rat calvarial osteoblasts was reduced by 75% at 35.5°C and by 95% at 34°C after 14-16 days culture, compared to 37°C. In addition to reductions in osteoblast cell number, expression of mRNAs for Runx2, alkaline phosphatase, osteocalcin and type I collagen were also down-regulated in hypothermia. In contrast, formation of osteoclasts in mononuclear cell cultures derived from mouse marrow, showed a 1.5 to 2-fold stimulation in hypothermia; resorption pit formation was similarly increased. Taken together, these data show that hypothermia exerts reciprocal effects on bone cell function by retarding osteoblast differentiation and bone formation, whilst increasing osteoclastogenesis and thus resorption. These results suggest the possibility that hypothermia in the elderly could potentially have a direct, negative impact on bone metabolism.     

Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PP_i). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2P_i). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PP_i-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.     

Serotonergic 5-HT2B Receptor Controls Tissue-nonspecific Alkaline Phosphatase Activity in Osteoblasts via Eicosanoids and Phosphatidylinositol-specific Phospholipase C

In previous studies, we observed that mice knocked out for the serotonin-2B receptor (5-HT_2BR) show defects in bone homeostasis. The present work focuses on the downstream targets relaying the anabolic function of this receptor in osteoblasts. A functional link between the 5-HT_2BR and the activity of the tissue-nonspecific alkaline phosphatase (TNAP) is established using the C1 osteoprogenitor cell line. During C1 osteogenic differentiation, both 5-HT_2BR and TNAP mRNA translations are delayed with respect to extracellular matrix deposition. Once the receptor is expressed, it constitutively controls TNAP activity at a post-translational level along the overall period of mineral deposition. Indeed, pharmacological inhibition of the 5-HT_2BR intrinsic activity or shRNA-mediated 5-HT_2BR knockdown prevents TNAP activation, but not its mRNA translation. In contrast, agonist stimulation of the receptor further increases TNAP activity during the initial mineralization phase. Building upon our previous observations that the 5-HT_2BR couples with the phospholipase A2 pathway and prostaglandin production at the beginning of mineral deposition, we show that the 5-HT_2BR controls leukotriene synthesis via phospholipase A2 at the terminal stages of C1 differentiation. These two 5-HT_2BR-dependent eicosanoid productions delineate distinct time windows of TNAP regulation during the osteogenic program. Finally, prostaglandins or leukotrienes are shown to relay the post-translational activation of TNAP via stimulation of the phosphatidylinositol-specific phospholipase C. In agreement with the above findings, primary calvarial osteoblasts from 5-HT_2BR-null mice exhibit defects in TNAP activity.     

Lipid links to better bone: a hypothesis

The combined resorptive activity of osteoclasts and the bone-generating function of osteoblasts result in the constant renewal of this vital tissue. It has long been appreciated that the coupled degradation and formation of bone is coordinately regulated by a complex interplay between endocrine and paracrine effectors; a recent report by now documents the possibility that cannabinoid receptors may also impact bone mass.     

The Roles of P2Y2 Purinergic Receptors in Osteoblasts and Mechanotransduction

We previously demonstrated, using osteoblastic MC3T3-E1 cells, that P2Y₂ purinergic receptors are involved in osteoblast mechanotransduction. In this study, our objective was to further investigate, using a knockout mouse model, the roles of P2Y₂ receptors in bone mechanobiology. We first examined bone structure with micro-CT and measured bone mechanical properties with three point bending experiments in both wild type mice and P2Y₂ knockout mice. We found that bones from P2Y₂ knockout mice have significantly decreased bone volume, bone thickness, bone stiffness and bone ultimate breaking force at 17 week old age. In order to elucidate the mechanisms by which P2Y₂ receptors contribute to bone biology, we examined differentiation and mineralization of bone marrow cells from wild type and P2Y₂ knockout mice. We found that P2Y₂ receptor deficiency reduces the differentiation and mineralization of bone marrow cells. Next, we compared the response of primary osteoblasts, from both wild type and P2Y₂ knockout mice, to ATP and mechanical stimulation (oscillatory fluid flow), and found that osteoblasts from wild type mice have a stronger response, in terms of ERK1/2 phosphorylation, to both ATP and fluid flow, relative to P2Y₂ knockout mice. However, we did not detect any difference in ATP release in response to fluid flow between wild type and P2Y₂ knock out osteoblasts. Our findings suggest that P2Y₂ receptors play important roles in bone marrow cell differentiation and mineralization as well as in bone cell mechanotransduction, leading to an osteopenic phenotype in P2Y₂ knockout mice.     

Expression and function of P2 receptors in bone

ATP (adenosine 5'-triphosphate) is one of the most important extracellular regulatory molecules in the skeleton. Extracellular ATP and other nucleotides signal through P2 receptors, a diverse group of receptors that are widely expressed by bone cells. P2 receptors are divided into two subclasses; P2Y G-protein coupled receptors, and P2X ligand-gated ion channels, and there is functional and molecular evidence for the expression of these receptors on both osteoblasts and osteoclasts. In order to activate P2 receptors, nucleotides must be released into the bone microenvironment. ATP is present in mmol concentrations in cells and can be released by cell lysis, cell trauma or physiological mechanisms, possibly through ABC transporters. Following co-activation of P2Y and PTH1 receptors on osteoblasts, there are multiple levels of interaction in downstream signalling that eventually lead to synergistic expression of osteoblastic genes, providing a mechanism for integrating local and systemic regulatory signals in bone particularly with regard to the activation of bone remodelling. Activation of P2Y1 receptors on osteoblasts enhances expression of RANKL leading indirectly to an increase in osteoclast formation and resorption. Expression of P2X7 inducible pores on osteoclast precursor cell membranes allows fusion to form multinucleated osteoclasts and blockade of this receptor inhibits resorption. The capacity of extracellular nucleotides to provide a highly localized and transient signal coupled with the profound effects of P2 receptor activation on osteoblastic and osteoclastic cells and the synergistic interactions with systemic hormones, indicate that nucleotides have a strong influence over bone tissue growth and regeneration.