PPAR agonists modulate human osteoclast formation and activity in vitro.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear steroid hormone superfamily and exist in three isoforms: PPARalpha, beta and gamma, each with specific functions. In this study, we have investigated the expression of PPARs by human osteoclast precursors and osteoclasts generated in vitro. In addition, the effects of fibrates and isoform-specific PPAR agonists on osteoclast formation and resorption in vitro were determined. Human peripheral blood mononuclear cells (PBMCs) were stimulated with human recombinant RANKL and M-CSF to generate osteoclasts. RNA was extracted at days 0, 7, 14 and 21 and RT-PCR for all three PPAR isoforms demonstrated their expression throughout this culture period. To determine the effect on osteoclast formation, PPAR agonists (10(-8) M to 10(-5) M) were added from the beginning of the culture until day 14 and the number of multinucleated osteoclasts counted. The effect of PPAR agonists on osteoclast function was similarly determined by treating mature, multinucleated osteoclasts cultured on dentine wafers with PPAR agonists (10(-8) M to 10(-5) M) for 7 days and quantifying resorption. Bezafibrate and fenofibrate, which non-discriminately activate all PPAR isoforms, significantly inhibited the formation of multinucleated osteoclasts from PBMC in vitro. Bezafibrate treatment of mature osteoclast resulted in 50% inhibition (at 10(-8) M and 10(-7) M) of resorption, yet fenofibrate had no significant effect. Activation of individual PPARs with isoform-specific agonist (GW9578, L165041 and ciglitizone which preferentially activate PPARalpha, beta and gamma respectively) resulted in significant dose-dependent inhibition of multinucleated osteoclast formation. Divergent effects on osteoclast resorption were observed; GW9578 had no significant effect on resorption, whereas ciglitizone and L165041 dose-dependently inhibited and stimulated resorption, respectively. These data show for the first time expression of all three PPAR isoforms throughout the development and maturation period of osteoclasts generated from human PBMCs. In addition, we demonstrate that isoform-specific PPAR agonists have strong effects on multinucleation and highly variable effects on bone resorption. In conclusion, this study highlights the potential of PPARs as therapeutic targets in diseases with accelerated osteoclast formation and resorption.

Multinucleated osteoclast formation in vivo and in vitro by P2X7 receptor-deficient mice.

The P2X7 receptor is a member of the family of P2X purinergic receptors, which upon sustained activation forms large pores in the plasma membrane. In cells of hematopoietic origin, P2X7 receptor activation has been shown to lead to multiple downstream events, including cytokine release, cell permeabilization, and apoptosis. This receptor has also been implicated in the generation of multinucleated giant cells, polykaryons, and osteoclasts. We have recently demonstrated that a blockade of this receptor inhibits osteoclast formation in vitro; therefore, we examined mice deficient in the P2X7 receptor in the context of bone. These mice were healthy and displayed no overt skeletal problems. Furthermore, we were able to demonstrate their ability to form multinucleated cells, in particular osteoclasts, both in vivo and in vitro. We also demonstrate the ability of P2X7R-/- multinucleated osteoclasts, upon stimulation with maitotoxin (MTX), to form pores in the plasma membrane in vitro. These findings are consistent with the existence of an endogenous pore structure present in osteoclast precursor cells that can be activated either by the P2X7 receptor, or in its absence, by alternative signals to mediate fusion and pore formation. These data provide further insight into the mode of action of the P2X7 receptor.

Release and interconversion of P2 receptor agonists by human osteoblast-like cells.

Nucleotides, acting as agonists at P2 receptors, are important extracellular signaling molecules in many tissues. In bone they affect both bone-forming osteoblast and bone-resorbing osteoclast cell activity. The presence of nucleotides in the extracellular microenvironment is largely determined by their release from cells and metabolism by ecto-enzymes, both of which have scarcely been studied in bone. We have investigated adenosine 5'-triphosphate (ATP) release from SaOS-2 osteoblastic cells and the activities of cell surface ecto-enzymes on ATP metabolism. ATP, but not LDH, was detected in SaOS-2 cell conditioned medium, suggesting these cells were actively releasing ATP. Introduction of ADP resulted in increased ATP concentrations in the medium, which was found not to be receptor mediated. Nucleotide inhibition and substrate specificity studies revealed an ecto-nucleoside diphosphokinase (ecto-NDPK) was responsible for the ADP-->ATP conversion; PCR and immunocytochemistry confirmed its presence. Analysis of ATP metabolism over time demonstrated overall ATP degradation was increased by inhibiting ecto-NDPK activity; confirming that the combined action of multiple osteoblast-expressed ecto-enzymes affected extracellular nucleotide concentration. The data establish the coexistence of ATP-consuming, and for the first time, ATP-generating activities on the osteoblast cell surface, the discovery of which has significant implications for studies involving P2 receptor subtypes in bone.

Blockade of the pore-forming P2X7 receptor inhibits formation of multinucleated human osteoclasts in vitro.

Osteoclasts are large, multinucleated, terminally differentiated cells formed by the fusion of mononuclear hemopoietic precursors. Their function is the resorption of bone, which is an essential part of the growth, modeling and remodeling of the skeleton. Though some osteoclast differentiation factors have recently been identified, the molecular basis for the fusion process that leads to multinucleation is poorly understood. The ATP-gated P2X7 receptor is a plasma membrane receptor belonging to the family of P2X purinergic receptors. It is known to be expressed by cells of hemopoietic origin where its activation leads to multiple downstream events including cytokine release, cell permeabilization and apoptosis. More recently this receptor has been implicated in the generation of multinucleated giant cells and polykaryons. Here we show that human osteoclasts express P2X7 receptors in vitro and in vivo, and that these receptors are functional in vitro, as assessed by pore-formation studies. More importantly, blockade of the P2X7 receptor with the antagonist oxidized ATP or a blocking monoclonal antibody significantly inhibits the fusion of osteoclast precursors to form multinucleated osteoclasts. Taken in combination with previous results from our laboratory demonstrating P2X7 receptor-mediated apoptosis and inhibition of bone resorption in vitro, these data suggest an important role for the P2X7 receptor in the regulation of the osteoclast population. The P2X7 receptor provides a significant new target for modulating osteoclast function in diseases characterized by increased osteoclast number and excessive bone turnover.

Adenosine triphosphate stimulates human osteoclast activity via upregulation of osteoblast-expressed receptor activator of nuclear factor-kappa B ligand.

Nucleotides such as adenosine triphosphate (ATP) and uridine triphosphate (UTP) exist in the extracellular environment where they are agonists at P2 receptors. Both P2Y G-protein-coupled receptors and P2X ligand-gated ion channels are expressed by osteoblasts and osteoclasts, reflected in the diverse nucleotide-induced effects reported to occur in bone. Previous reports have implicated ATP as a proresorptive agent; however, these studies were unable to determine whether ATP mediated its actions directly on osteoclasts, or indirectly via osteoblasts. The development of techniques to generate human osteoclasts in vitro has allowed us to further investigate the intriguing role of extracellular nucleotides with regard to osteoclast activity. This study reports that nearly all P2-receptor-subtype mRNAs were expressed throughout human osteoclast development, and provides evidence for functional P2 receptor expression by these cells. In cultures of human osteoclasts alone, neither ATP nor UTP affected the quantity of resorption by these cells; however, in cocultures of osteoblast-like UMR-106 cells and human osteoclasts, ATP, but not UTP, greatly enhanced resorption, indicating a role for osteoblasts in mediating the proresorptive effects of ATP. Furthermore, ATP, but not UTP, elevated receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein expression by UMR-106 cells. These data are consistent with observations that UMR-106 cells predominantly express P2Y(1) with low expression of P2Y(2), thereby explaining the response to ATP and not UTP, and further substantiating the involvement of osteoblasts in ATP-induced effects on osteoclasts. These results significantly advance our understanding of the role of P2 receptors in bone, and indicate that local-acting ATP may play a pivotal role in osteoclast activation at bone-resorbing sites by inducing elevated expression of RANKL.

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.

Extracellular nucleotide signaling: a mechanism for integrating local and systemic responses in the activation of bone remodeling.

Bone turnover occurs at discreet sites in the remodeling skeleton. The focal nature of this process indicates that local cues may facilitate the activation of bone cells by systemic factors. Nucleotides such as adenosine triphosphate (ATP) are locally released, short-lived, yet potent extracellular signaling molecules. These ligands act at a large family of receptors-the P2 receptors, which are subdivided into P2Y and P2X subtypes based on mechanism of signal transduction. Nucleotides enter the extracellular milieu via non-lytic and lytic mechanisms where they activate multiple P2 receptor types expressed by both osteoblasts and osteoclasts. In this review the release of ATP by bone cells is discussed in the context of activation of bone remodeling. We provide compelling evidence that nucleotides, acting via P2Y receptors, are potent potentiators of parathyroid hormone-induced signaling and transcriptional activation in osteoblasts. The provision of a mechanism to induce activation of osteoblasts above a threshold attained by systemic factors alone may facilitate focal remodeling and address the paradox of why systemic regulators like PTH exert effects at discreet sites.

Parathyroid hormone potentiates nucleotide-induced [Ca2+]i release in rat osteoblasts independently of Gq activation or cyclic monophosphate accumulation. A mechanism for localizing systemic responses in bone.

The regulation of tissue turnover requires the coordinated activity of both local and systemic factors. Nucleotides exist transiently in the extracellular environment, where they serve as ligands to P2 receptors. Here we report that the localized release of these nucleotides can sensitize osteoblasts to the activity of systemic factors. We have investigated the ability of parathyroid hormone (PTH), a principal regulator of bone resorption and formation, to potentiate signals arising from nucleotide stimulation of UMR-106 clonal rat osteoblasts. PTH receptor activation alone did not lead to [Ca(2+)](i) elevation in these cells, indicating no G(q) coupling, however, activation of G(q)-coupled P2Y(1) receptors resulted in characteristic [Ca(2+)](i) release. PTH potentiated this nucleotide-induced Ca(2+) release, independently of Ca(2+) influx. PTH-(1-31), which activates only G(s), mimicked the actions of PTH-(1-34), whereas PTH-(3-34), which only activates G(q), was unable to potentiate nucleotide-induced [Ca(2+)](i) release. Despite this coupling of the PTHR to G(s), cAMP accumulation or protein kinase A activation did not contribute to the potentiation. 3-Isobutyl-1-methylxanthine, but not forskolin effectively potentiated nucleotide-induced [Ca(2+)](i) release, however, further experiments proved that cyclic monophosphates were not involved in the potentiation mechanism. Costimulation of UMR-106 cells with P2Y(1) agonists and PTH led to increased levels of cAMP response element-binding protein phosphorylation and a synergistic effect was observed on endogenous c-fos gene expression following costimulation. In fact the calcium responsive Ca/cAMP response element of the c-fos promoter alone was effective at driving this synergistic gene expression. These findings demonstrate that nucleotides can provide a targeted response to systemic factors, such as PTH, and have important implications for PTH-induced signaling in bone.