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Biomedical subjects

J A Gallagher

Publications and source records attributed to J A Gallagher.

At least 19 recordsLinked to original sources
PubMed2006

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.

Acetates↗
PubMed2004

Knock-in of nuclear localised beta-galactosidase reveals that the tyrosine phosphatase Ptprv is specifically expressed in cells of the bone collar.

Ptprv is a member of the transmembrane tyrosine phosphatase gene family reported to be expressed in osteoblasts and gonads. To better define the developmental and tissue specificity of Ptprv expression, we generated knock-in mice expressing a nuclear localised beta-galactosidase reporter under the control of resident Ptprv regulatory elements. Histochemical staining of Ptprv-nLacZ mice revealed that Ptprv expression is readily detectable in the foetal gonadal ridge of both sexes and in adult gonads where it is localised to Sertoli cells of the testis and celomic epithelial cells of the ovaries. During early limb development, Ptprv expression is prominent in the apical ectodermal ridge of the limb bud. At latter stages of development, Ptprv is predominantly expressed in the perichondrial and periosteal region of long bones, known as the bone collar. In contrast to previous indications from in vitro studies, there is little if any expression in mature osteoblasts in vivo. Analysis of Ptprv mRNA localisation by in situ hybridization in parallel with molecular markers of chondrocytes and osteoblasts confirmed the specific expression of Ptprv in immature bone collar cells. The specificity of Ptprv expression in these cells may be a useful tool to elucidate their role in the transition of skeletal elements from cartilage template to bone.

Animals↗
PubMed2004

Cloning and characterization of a putative fructosyltransferase and two putative invertase genes from the temperate grass Lolium temulentum L.

The invertases of Lolium temulentum have been characterized at the enzyme level. However, studies on the expression of the genes coding for these enzymes have been lacking. To elucidate the role of acid 'invertase-like' genes in sucrose metabolism and carbon partitioning in Gramineae further, three 'invertase-like' homologous clones were isolated from L. temulentum cDNA expression libraries based on leaf tissue, using maize soluble invertase probes. The effect of developmental stage and alterations in carbohydrate status on the expression and tissue distribution of these genes was investigated. The three highly homologous genes (Inv 1:2, Inv 1:4, and FT 2:2) show different patterns of expression and different tissue distribution. Inv 1:2 was predominantly expressed in root tissue. Expression increased during the dark in root and tiller base tissue. Minimal variations in gene expression were observed in leaf tissue following changes in carbohydrate status. Inv 1:4 was predominantly expressed in tiller bases, leaf sheath, and leaf base, with increased expression in tissue samples in the dark period. FT 2:2 was also predominantly expressed in tiller bases, leaf sheath, and leaf base. Higher expression was observed in leaf tissue following increases in carbohydrate content, in a manner that paralleled the regulation and spatial occurrence of fructan in the leaf tissue. Whilst invertases and fructosyltransferases are difficult to distinguish at the level of the whole sequence, analysis of 5' sequence and specific amino acids allows discrimination which correlates with patterns of expression within the tissue. Based on expression patterns and sequence characteristics, it is proposed that Inv 1:2 and Inv 1:4 code for soluble acid invertases, whilst FT 2:2 codes for a fructosyltransferase.

Amino Acid Sequence↗
PubMed2004

ATP P2 receptors and regulation of bone effector cells.

ATP, signaling through P2 receptors, is one of the most important extracellular regulatory molecules in the skeleton. P2 receptors are divided into two subclasses, P2Y which are G-protein coupled and P2X which are ligand-gated ion channels. There is molecular and functional evidence for widespread expression of both subclasses of receptors by bone cells. Co-activation of P2Y and PTH1 receptors on osteoblasts, leads to synergistic expression of osteoblastic genes, providing a mechanism for integrating local and systemic regulatory signals in bone. 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. Bone cells release nucleotides into the extracellular environment to provide highly localized and transient signals that regulate bone formation and bone resorption.

Adenosine Triphosphate↗
PubMed2003

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↗
PubMed2003

Quantitative evaluation of the biocompatible and osteogenic properties of a range of biphasic calcium phosphate (BCP) granules using primary cultures of human osteoblasts and monocytes.

A range of 50% porous gamma-sterilized biphasic calcium phosphate (BCP) granules, (20, 50, 80, and 100% tricalcium phosphate, TCP) were classified into two distinct size ranges, small 2-4 mm in diameter and large 4-6 mm in diameter, and their potential as bone graft extender materials was assessed in vitro using culture systems of primary-derived peripheral human blood monocytes and human osteoblasts isolated from bone. The effect of the in vitro culture conditions was evaluated prior to the introduction of the test substrates. The cellular response was assessed via quantification of viable cell adhesion to the materials, lactate dehydrogenase (LDH), the production and release of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and prostaglandin E2 (PGE2). The higher content TCP materials, 80% and 100% TCP, had a detrimental effect on viable cell adhesion after day 1, which was not related to calcium release from the granules within the local environment. TCP granules (20% and 50%) initiated a controlled level of inflammatory response that sustained and promoted viable macrophage adhesion throughout the test period, The percentage of TCP within the BCP granules was a governing factor in determining the cellular response.

Biocompatible Materials↗
PubMed2003

Quantitative assessment of the response of primary derived human osteoblasts and macrophages to a range of nanotopography surfaces in a single culture model in vitro.

The effect of nanotopography on a range of Ti oxide surfaces was determined. Flat Ti, 3%, 19%, 30% and 43% topography densities of 110 nm high hemispherical protrusions were cultured in contact with primary derived human macrophages and osteoblasts in single culture models. Prior to introduction of the test substrate the phenotype and optimum conditions for in vitro cell culture were established. The cellular response was investigated and quantified by assessments of cytoskeletal development and orientation, viable cell adhesion, cytokine production and release and RT-PCR analysis of osteogenic markers. The tested nanotopographies did not have a statistically significant effect on viable cell adhesion and subsequent cytoskeletal formation. Surface chemistry was the dominant factor as established via incorporation of a tissue culture polystyrene, TCPS, control. The topography surfaces induced a release of chemotactic macrophage activation agents at 1 day in conjunction with stress fibre formation and a subsequent fibronectin network formation. Osteoblasts migrated away from the topography surfaces to the exposed TCPS within the wells during the 7-day period.

Cell Adhesion↗
PubMed2003

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.

Adenosine Diphosphate↗
PubMed2003

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.

Animals↗
PubMed2002

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.

Adenosine Triphosphate↗
PubMed2002

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.

Journal Article↗
PubMed2001

Novel synthesis of bis(phosphonic acid)-steroid conjugates.

An efficient synthesis has been realized for several members of a new class of potential bone resorption inhibitors consisting of steroidal oestrogenic units linked at the 3 and 17 positions to a geminal bisphosphonate moiety through an ester linkage of variable length. The convergent synthesis utilizes benzyl bisphosphonates, transesterification, and Meldrum's acid chemistry and has the potential to allow many oestrogenic derivatives as well as other biologically active compounds to be coupled to the geminal bisphosphonate moeity.

Bone Resorption↗
PubMed2001

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.

Adenosine Triphosphate↗
PubMed2001

Expression of a P2X7 receptor by a subpopulation of human osteoblasts.

There is now conclusive evidence that extracellular nucleotides acting via cell surface P2 receptors are important local modulators of bone cell function. Multiple subtypes of P2 receptors have been localized to bone, where their activation modulates multiple processes including osteoblast proliferation, osteoblast-mediated bone formation, and osteoclast formation and resorptive capacity. Locally released nucleotides also have been shown to sensitize surrounding cells to the action of systemic factors such as parathyroid hormone (PTH). In nonskeletal tissue recent attention has focused on one particular P2 receptor, the P2X7 receptor (previously termed P2Z), and its ability to form nonselective aqueous pores in the plasma membrane on prolonged stimulation. Expression of this receptor originally was thought to be restricted to cells of hemopoietic origin, in which it has been implicated in cell fusion, apoptosis, and release of proinflammatory cytokines. However, recent reports have indicated expression of this receptor in cells of stromal origin. In this study, we investigated the expression of the P2X7 receptor in two human osteosarcoma cell lines, as well as several populations of primary human bone-derived cells (HBDCs) at the levels of messenger RNA (mRNA) and protein. We found that there is a subpopulation of osteoblasts that expresses the P2X7 receptor and that these receptors are functional as assessed by monitoring ethidium bromide uptake following pore formation. Inhibition of delayed lactate dehydrogenase (LDH) release in response to the specific agonist 2',3'-(4-benzoyl)-benzoyl-adenosine triphosphate (BzATP) by the nonspecific P2X receptor antagonist PPADS confirmed a receptor-mediated event. After treatment with BzATP SaOS-2 cells exhibited dramatic morphological changes consistent with those observed after P2X7-mediated apoptosis in hemopoietic cells. Dual staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) and a P2X7-specific monoclonal antibody confirmed the induction of apoptosis in osteoblasts expressing the P2X7 receptor. These data show for the first time the expression of functional P2X7 receptors in a subpopulation of osteoblasts, activation of which can result in ATP-mediated apoptosis.

Adenosine Triphosphate↗
PubMed2000

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.

1-Methyl-3-isobutylxanthine↗
PubMed2000

A CD36-binding peptide from thrombospondin-1 can stimulate resorption by osteoclasts in vitro.

Thrombospondin-1 (TSP-1), purified from platelets, stimulates resorption by avian osteoclasts in an in vitro resorption assay. TSP-1 binds to a number of different cellular receptors via different domains of the molecule and several short receptor-binding sequences have been identified within the TSP-1 molecule. In this study, we have used synthetic peptides representing these various sequences in order to identify the cellular receptor and TSP domain responsible for stimulation of resorption. We show that one peptide CSVTCG, which represents the CD36-binding region of TSP-1, stimulates resorption in a fashion similar to the intact molecule, while the peptides RGDS, RFYVVMWK, and RFYVVM, representing other cell-binding domains of TSP, have no effect on resorption. Using RT-PCR and immunoblotting, we further demonstrate expression of CD36 in human osteoclastoma (giant cell tumour), primary human bone derived cells, and clonal osteoblastic cells. These studies suggest that CD36 is involved in regulation of resorption by osteoclasts and is the receptor responsible for the resorption-promoting effects of TSP-1.

Amino Acid Sequence↗
PubMed2000

Absence of measles virus and canine distemper virus transcripts in long-term bone marrow cultures from patients with Paget's disease of bone.

The presence of intranuclear inclusions in pagetic osteoclasts with similar characteristics to those seen in some slow viral diseases has lead to the hypothesis that Paget's disease is caused by a similar infection. Bone marrow aspirates from seven patients with hemipelvic Paget's disease were taken from both sides of the pelvis and cultured under identical conditions. RNA was extracted after approximately 2 weeks of culture and reverse transcriptase-linked polymerase chain reaction used to investigate the expression of measles and canine distemper virus RNA. We were unable to detect the presence of measles virus (MV) or canine distemper virus (CDV) transcripts in bone marrow cultures from either affected or unaffected sites in any of our patients. The results of this study do not support a role for MV or CDV in the pathogenesis of Paget's disease of bone.

Animals↗