Katherine A. Buckley

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.

Human Keratinocytes Release ATP and Utilize Three Mechanisms for Nucleotide Interconversion at the Cell Surface

Nucleotide activation of P2 receptors is important in autocrine and paracrine regulation in many tissues. In the epidermis, nucleotides are involved in proliferation, differentiation, and apoptosis. In this study, we have used a combination of luciferin-luciferase luminometry, pharmacological inhibitors, and confocal microscopy to demonstrate that HaCaT keratinocytes release ATP into the culture medium, and that there are three mechanisms for nucleotide interconversion, resulting in ATP generation at the cell surface. Addition of ADP, GTP, or UTP to culture medium elevated the ATP concentration. ADP to ATP conversion was inhibited by diadenosine pentaphosphate, oligomycin, and UDP, suggesting the involvement of cell surface adenylate kinase, F1F0 ATP synthase, and nucleoside diphosphokinase (NDPK), respectively, which was supported by immunohistochemistry. Simultaneous addition of ADP and GTP elevated ATP above that for each nucleotide alone indicating that GTP acts as a phosphate donor. However, the activity of NDPK, F1F0 ATP synthase or the forward reaction of adenylate kinase could not fully account for the culture medium ATP content. We postulate that this discrepancy is due to the reverse reaction of adenylate kinase utilizing AMP. In normal human skin, F1F0 ATP synthase and NDPK were differentially localized, with mitochondrial expression in the basal layer, and cell surface expression in the differentiated layers. We and others have previously demonstrated that keratinocytes express multiple P2 receptors. In this study we now identify the potential sources of extracellular ATP required to activate these receptors and provide better understanding of the role of nucleotides in normal epidermal homeostasis and wound healing.

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

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.

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. FASEB J. 17, 1401–1410 (2003)

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 [Ca2+] i elevation in these cells, indicating no Gq coupling, however, activation of Gq-coupled P2Y1 receptors resulted in characteristic [Ca2+] i release. PTH potentiated this nucleotide-induced Ca2+ release, independently of Ca2+ influx. PTH-(1–31), which activates only Gs, mimicked the actions of PTH-(1–34), whereas PTH-(3–34), which only activates Gq, was unable to potentiate nucleotide-induced [Ca2+] i release. Despite this coupling of the PTHR to Gs, cAMP accumulation or protein kinase A activation did not contribute to the potentiation. 3-Isobutyl-1-methylxanthine, but not forskolin effectively potentiated nucleotide-induced [Ca2+] i release, however, further experiments proved that cyclic monophosphates were not involved in the potentiation mechanism. Costimulation of UMR-106 cells with P2Y1agonists 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.

Receptor activator for nuclear factor kappaB ligand and osteoprotegerin: regulators of bone physiology and immune responses/potential therapeutic agents and biochemical markers.

The discovery of receptor activator for nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) as the fundamental factors in controlling osteoclast formation and activation has led to a greater understanding of bone biology over the past few years. Here we discuss the role of these molecules in immunology and skeletal remodelling and assess their involvement in diseases of bones and joints, including rheumatoid arthritis, Pagets disease, post-menopausal osteoporosis and malignant bone diseases. OPG has been identified as a potential anabolic agent for treating conditions in which there is net bone loss and is currently in Phase I clinical trials. This review examines the current evidence indicating that OPG increases bone mass, and discusses other possible beneficial effects of OPG, such as inhibition of tumour growth and relief from bone cancer pain. OPG can be measured in human serum, and numerous studies have suggested that increased or decreased serum concentrations of this molecule can indicate the existence of remodelling disorders. Here we discuss how abnomnal serum OPG concentrations could potentially be used to indicate imbalances of bone resorption and formation. The possible applications of serum OPG concentration as a marker for non-skeletal disease conditions are also considered.

The effects of P2X7 receptor antagonists on the formation and function of human osteoclasts in vitro.

The P2X7 receptor (P2X7R) has been implicated in the process of multinucleation and cell fusion. We have previously demonstrated that blockade of P2X7Rs on osteoclast precursors using a blocking antibody inhibited multinucleated osteoclast formation in vitro, but that P2X7R KO mice maintain the ability to form multinucleated osteoclasts. This apparent contradiction of the role the P2X7R plays in multinucleation has prompted us to examine the effect of the most commonly used and recently available P2X7R antagonists on osteoclast formation and function. When added to recombinant RANKL and M-CSF human blood monocytes cultures, all but one compound, decreased the formation and function of multinucleated TRAP-positive osteoclasts in a concentration-dependent manner. These data provide further evidence for the role of the P2X7R in the formation of functional human multinucleated osteoclasts and highlight the importance of selection of antagonists for use in long-term experiments.

Human osteoclast culture from peripheral blood monocytes: phenotypic characterization and quantitation of resorption.

1 Establishment and Maintenance of Normal Human Keratinocyte Cultures Claire Linge ............................................................................................ 1 2 Cultivation of Normal Human Epidermal Melanocytes in the Absence of Phorbol Esters Mei-Yu Hsu, Ling Li, and Meenhard Herlyn ...................................... 13 3 Isolation and Culture of Human Osteoblasts Alison Gartland, Katherine A. Buckley, Jane P. Dillon, Judith M. Curran, John A. Hunt, and James A. Gallagher ......... 29 4 Human Osteoclast Culture from Peripheral Blood Monocytes: Phenotypic Characterization and Quantitation of Resorption Katherine A. Buckley, Benjamin Y. Y. Chan, William D. Fraser, and James A. Gallager .................................................................... 55 5 Human Chondrocyte Cultures as Models of Cartilage-Specific Gene Regulation Mary B. Goldring .................................................................................. 69 6 Human Myoblasts and Muscle-Derived SP Cells Grace K. Pavlath and Emanuela Gussoni ........................................ 97 7 Cell Cultures of Autopsy-Derived Fibroblasts Volker Meske, Frank Albert, and Thomas G. Ohm ........................ 111 8 Primary Culture and Differentiation of Human Adipocyte Precursor Cells Vanessa van Harmelen, Thomas Skurk, and Hans Hauner ......... 125 9 Human Mononuclear Phagocytes in Tissue Culture Yona Keisari ....................................................................................... 137 10 Purification of Peripheral Blood Natural Killer Cells Bice Perussia and Matthew J. Loza ................................................ 147 11 Human Fetal Brain Cell Culture Mark P. Mattson ................................................................................. 163