Rheumatol Int. 2008 Dec;29(2):167-71.
Cyclic adenosine 5′-monophosphate in synovial fluid of rheumatoid arthritis and osteoarthritis patients.
Morovic-Vergles J, Culo MI, Gamulin S, Culo F.
Source
Division of Clinical Immunology and Rheumatology, Department of Internal Medicine, Dubrava University Hospital, and Department of Pathophysiology, Medical School University of Zagreb, Av. G. Suska 6, Zagreb, 10040, Croatia. jmorovic@kbd.hr
Abstract
The relationship between synovial fluid (SF) cAMP level and IL-18 and PGE2 SF levels in rheumatoid arthritis (RA) and osteoarthritis (OA) patients, and between SF cAMP level and disease as well as inflammatory activity in RA were investigated in 17 RA and 19 OA patients. Erythrocyte sedimentation rate (ESR), serum (S) C-reactive protein (CRP) level and SF IL-18 level were higher in RA than in OA patients. SF PGE2 level was similar in both groups. SF cAMP level was higher in OA than in RA patients. In RA patients, SF cAMP level showed negative correlation with Disease Activity Score including a 28-joint count and S CRP, ESR and SF IL-18 level.
The results suggest that cAMP promotes anti-inflammatory response in RA and OA patients, which is higher in the latter.
Promotion of anti-inflammatory response by cAMP elevating agents might be useful in the treatment of RA.
PMID:18695981[PubMed – indexed for MEDLINE]

Br J Pharmacol. 2010 May;160(1):101-15.
Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes.
Varani K, Vincenzi F, Tosi A, Targa M, Masieri FF, Ongaro A, De Mattei M, Massari L, Borea PA.
Source
Department of Clinical and Experimental Medicine, University of Ferrara, Ferrara, Italy.
Abstract
BACKGROUND AND PURPOSE:
Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A(1), A(2A), A(2B) and A(3)) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail.
We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes.
EXPERIMENTAL APPROACH:
mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-kappaB activation, tumour necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis.
KEY RESULTS:
mRNA and protein for A(1), A(2A), A(2B) and A(3) adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A(2A) and A(3) adenosine receptors inhibited p38 MAPK and NF-kappaB pathways, an effect abolished by selective adenosine antagonists. A(2A) and A(3) receptor agonists decreased TNF-alpha and IL-8 production. The phosphoinositide 3-kinase or G(s) pathways were involved in the functional responses of A(3) or A(2A) adenosine receptors.
Synoviocyte A(1) and A(2B) adenosine receptors were not implicated in the inflammatory process whereas stimulation of A(2A) and A(3) adenosine receptors was closely associated with a down-regulation of the inflammatory status.
CONCLUSIONS AND IMPLICATIONS:
These results indicate that A(2A) and A(3) adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation.

Front Biosci (Elite Ed). 2011 Jun 1;3:888-95.
The roles of adenosine and adenosine receptors in bone remodeling.
He W, Cronstein B.
Source
Division of Clinical Pharmacology, NYU School of Medicine, NYC, NY, NY 10016, USA.
Abstract
Adenosine regulates a wide variety of physiological processes including heart rate, vasodilation and inflammation through the activation of specific cell surface adenosine receptors. In addition to these well-established roles of adenosine, recent genetic and pharmacological research has implicated adenosine as an important regulator in bone remodeling.
The secretion of adenosine and the presence of its four receptors in bone cells have been well documented. More recently, we provided the first evidence that adenosine regulates osteoclast formation and function through A1 receptor (A1R), and showed that A1R-knockout mice have significantly increased bone volume as a result of impaired osteoclast-mediated bone resorption. Moreover, adenosine A1R-knockout mice are protective from boss loss following ovariectomy further supporting the involvement of adenosine in osteoclast formation and function.
This short review summarizes current knowledge related to the roles of adenosine and adenosine receptors in bone formation and remodeling.
A deeper insight into the regulation of bone metabolism by adenosine receptors should assist in developing new therapies for osteoporosis.
PMID:21622100[PubMed – indexed for MEDLINE]

Sci Transl Med. 2012 May 23;4(135):135ra65.
Adenosine A2A receptor activation prevents wear particle-induced osteolysis.
Mediero A, Frenkel SR, Wilder T, He W, Mazumder A, Cronstein BN.
Source
Division of Translational Medicine, Department of Medicine, New York University School of Medicine, New York, NY 10016, USA.
Abstract
Prosthesis loosening, associated with wear particle-induced inflammation and osteoclast-mediated bone destruction, is a common cause for joint implant failure, leading to revision surgery. Adenosine A(2A) receptors (A(2A)Rs) mediate potent anti-inflammatory effects in many tissues and prevent osteoclast differentiation.
We tested the hypothesis that an A(2A)R agonist could reduce osteoclast-mediated bone resorption in a murine calvaria model of wear particle-induced bone resorption. C57BL/6 and A(2A)R knockout (A(2A)R KO) mice received ultrahigh-molecular weight polyethylene particles and were treated daily with either saline or the A(2A)R agonist CGS21680. After 2 weeks, micro-computed tomography of calvaria demonstrated that CGS21680 reduced particle-induced bone pitting and porosity in a dose-dependent manner, increasing cortical bone and bone volume compared to control mice. Histological examination demonstrated diminished inflammation after treatment with CGS21680. In A(2A)R KO mice, CGS21680 did not affect osteoclast-mediated bone resorption or inflammation. Levels of bone resorption markers receptor activator of nuclear factor κB (RANK), RANK ligand, cathepsin K, CD163, and osteopontin were reduced after CGS21680 treatment, together with a reduction in osteoclasts. Secretion of interleukin-1β (IL-1β) and tumor necrosis factor-α was significantly decreased, whereas IL-10 was markedly increased in bone by CGS21680. These results in mice suggest that site-specific delivery of an adenosine A(2A)R agonist could enhance implant survival, delaying or eliminating the need for revision arthroplastic surgery.
PMID:22623741[PubMed – indexed for MEDLINE] PMCID:PMC3386559

Calcif Tissue Int. 1996 Feb;58(2):109-13.
ATP and adenosine act as a mitogen for osteoblast-like cells (MC3T3-E1).
Shimegi S.
Source
Faculty of Health and Sport Sciences, Osaka University, Japan.
Abstract
Extracellular ATP, and to a lesser extent adenosine, an ATP metabolite, stimulated cell proliferation in osteoblast-like cells (MC3T3-E1).
ATP increased cytosolic Ca2+ due to Ca2+ mobilization from intracellular storage in the same concentration range of the nucleotide as that effective for DNA synthesis, suggesting the mediation of the phospholipase C/Ca2+ system in the mitogenic action.
Since adenosine induced no Ca2+ mobilization, P2-purinergic receptor appears to be associated with ATP actions.
The growth-promoting effect of ATP was not inhibited by H7, a protein kinase C inhibitor, and indomethacin, a cyclooxygenase inhibitor, indicating no involvement of activation of protein kinase C and production of prostaglandins in ATP-induced mitogenic signals.
Either ATP or adenosine remarkably and synergistically potentiated platelet derived growth factor-induced DNA synthesis.
These findings suggest that extracellular ATP and adenosine may play a physiological role in the regulation of bone formation.
PMID:8998680[PubMed – indexed for MEDLINE]

Am J Physiol Cell Physiol. 2000 Aug;279(2):C510-9.
ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells.
Nakamura E, Uezono Y, Narusawa K, Shibuya I, Oishi Y, Tanaka M, Yanagihara N, Nakamura T, Izumi F.
Source
Department of Orthopedic Surgery, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan.
Abstract
In human osteoblast-like MG-63 cells, extracellular ATP increased [(3)H]thymidine incorporation and cell proliferation and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced [(3)H]thymidine incorporation. ATP-induced [(3)H]thymidine incorporation was mimicked by the nonhydrolyzable ATP analogs adenosine 5′-O-(3-thiotriphosphate) and adenosine 5′-adenylylimidodiphosphate and was inhibited by the P2 purinoceptor antagonist suramin, suggesting involvement of P2 purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptor antagonist reactive blue 2 did not affect [(3)H]thymidine incorporation, whereas the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4-disulfonic acid inhibited ATP-induced [(3)H]thymidine incorporation, suggesting that ATP-induced DNA synthesis was mediated by P2X receptors. RT-PCR analysis revealed that MG-63 cells expressed P2X(4), P2X(5), P2X(6), and P2X(7), but not P2X(1), P2X(2), and P2X(3), receptors. In fura 2-loaded cells, not only ATP, but also UTP, increased intracellular Ca(2+) concentration, and inhibitors for several Ca(2+)-activated protein kinases had no effect on ATP-induced DNA synthesis, suggesting that an increase in intracellular Ca(2+) concentration is not indispensable for ATP-induced DNA synthesis. ATP increased mitogen-activated protein kinase activity in a Ca(2+)-independent manner and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced kinase activity. Furthermore, the mitogen-activated protein kinase kinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis.
We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors by activating a mitogen-activated protein kinase pathway.
PMID:10913018[PubMed – indexed for MEDLINE]

Tissue Eng Part A. 2013 Oct;19(19-20):2188-200.
Supplementation of Exogenous Adenosine 5′-Triphosphate Enhances Mechanical Properties of 3D Cell-Agarose Constructs for Cartilage Tissue Engineering.
Gadjanski I, Yodmuang S, Spiller K, Bhumiratana S, Vunjak-Novakovic G.
Source
1 Department of Biomedical Engineering, Columbia University , New York, New York.
Abstract
Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation.
However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation.
In particular, the purinergic, adenosine 5′-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage.
We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel.
For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation.
Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content.
Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II.
These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.
PMID:23651296[PubMed – in process] PMCID:PMC3761430[Available on 2014/10/1]

Bone. 2001 May;28(5):507-12.
Extracellular nucleotide signaling: a mechanism for integrating local and systemic responses in the activation of bone remodeling.
Bowler WB, Buckley KA, Gartland A, Hipskind RA, Bilbe G, Gallagher JA.
Source
Human Bone Cell Research Group, Department of Human Anatomy & Cell Biology, University of Liverpool, Liverpool L69 3GE, UK. wbb@liv.ac.uk
Abstract
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.
PMID:11344050[PubMed – indexed for MEDLINE]

Bone. 2006 Aug;39(2):300-9. Epub 2006 Apr 17.
Osteoblast responses to nucleotides increase during differentiation.
Orriss IR, Knight GE, Ranasinghe S, Burnstock G, Arnett TR.
Source Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK.
Abstract
Accumulating evidence suggests that extracellular nucleotides, signaling through P2 receptors, play a role in modulating bone cell function.
ATP and ADP stimulate osteoclastic resorption, while ATP and UTP are powerful inhibitors of bone formation by osteoblasts. We investigated changes in the expression of P2 receptors with cell differentiation in primary osteoblast cultures. Rat calvarial osteoblasts, cultured for up to 10 days, were loaded with the intracellular Ca(2+)-sensing fluorophore, Fluo-4 AM, and a fluorescence imaging plate reader was used to measure responses to nucleotide agonists. Peak responses occurred within 20 s and were evoked by ATP or UTP at concentrations as low as 2 microM. Osteoblast number doubled between day 4 and 10 of culture, but the peak intracellular Ca(2+) response to ATP or UTP increased up to 6-fold over the same period, indicating that osteoblast responsiveness to nucleotides increases as cell differentiation proceeds. The approximate order of potency for the most active nucleotide agonists at day 8 of culture was ATP > UTP and ATPgammaS > ADP > UDP, consistent with the expression of functional P2Y(2), P2X(2), P2Y(4), P2Y(1) and P2Y(6) receptors. Smaller responses were elicited by 2-MeSATP, Bz-ATP and alpha,beta-meATP, additionally suggesting the presence of functional P2X(1), P2X(3), P2X(5) and P2X(7) receptors. Expression of mRNA for the ATP- and UTP-selective P2Y(2) receptor increased strongly between day 6 and 15 in primary rat osteoblasts, whereas mRNAs for the P2Y(4) (also ATP/UTP selective) and P2Y(6) (UDP/UTP selective) receptors were highly expressed at intermediate time points. In contrast, mRNA for the cell-proliferation-associated P2X(5) receptor decreased to undetectable as osteoblasts matured, but mRNA for the cell-death-associated P2X(7) receptor was detected at all time points. Similar trends were evident using immunostaining and Western blotting for P2 receptors. Exposure to 10 muM ATP or UTP during days 10-14 of culture was sufficient to cause near-total blockade of the ‘trabecular’ bone nodules formed by osteoblasts; however, UDP and ADP were without effect. Our results show that there is a shift from P2X to P2Y expression during differentiation in culture, with mature osteoblasts preferentially expressing the P2Y(2) receptor and to a lesser extent P2Y(4) and P2Y(6) receptors.
Taken together, these data suggest that the P2Y(2) receptor, and possibly the P2Y(4) receptor, could function as ‘off-switches’ for mineralized bone formation.
PMID:  16616882   [PubMed – indexed for MEDLINE]

Br J Pharmacol. 2013 Jul;169(6):1372-88.
Activation of adenosine A2A receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation.
Mediero A, Perez-Aso M, Cronstein BN.
Source
Department of Medicine, Division of Translational Medicine, NYU School of Medicine, New York, NY 10016, USA.
Abstract
BACKGROUND AND PURPOSE:
We previously reported that adenosine, acting at adenosine A2A receptors (A2A R), inhibits osteoclast (OC) differentiation in vitro (A2A R activation OC formation reduces by half) and in vivo.
For a better understanding how adenosine A2A R stimulation regulates OC differentiation, we dissected the signalling pathways involved in A2A R signalling.
EXPERIMENTAL APPROACH:
OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A2A R agonist CGS21680, the A2A R antagonist ZM241385, PKA activators (8-Cl-cAMP 100 nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR.
KEY RESULTS:
A2A R stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A2A R stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A2A R stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A2A R stimulation inhibited OC expression of differentiation markers by a PKA-mechanism.
CONCLUSIONS AND IMPLICATIONS:
A2A R activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like Rheumatoid Arthritis.
© 2013 The British Pharmacological Society.PMID:23647065[PubMed – in process]

Trends Endocrinol Metab. 2013 Jun;24(6):290-300
Adenosine and bone metabolism.
Mediero A, Cronstein BN.
Source
Department of Medicine, Division of Translational Medicine, New York University School of Medicine, 550 First Avenue, MSB251, New York, NY 10016, USA.
Abstract
Bone is a dynamic organ that undergoes continuous remodeling while maintaining a balance between bone formation and resorption. Osteoblasts, which synthesize and mineralize new bone, and osteoclasts, the cells that resorb bone, act in concert to maintain bone homeostasis.
In recent years, there has been increasing appreciation of purinergic regulation of bone metabolism. Adenosine, released locally, mediates its physiologic and pharmacologic actions via interactions with G protein-coupled receptors, and recent work has indicated that these receptors are involved in the regulation of osteoclast differentiation and function, as well as in osteoblast differentiation and bone formation.
Moreover, adenosine receptors also regulate chondrocyte and cartilage homeostasis.
These recent findings underscore the potential therapeutic importance of adenosine receptors in regulating bone physiology and pathology.
Copyright © 2013 Elsevier Ltd. All rights reserved.

Life Sci. 2003 Aug 29;73(15):1973-83.
Polydeoxyribonucleotide (PDRN) promotes human osteoblast proliferation: a new proposal for bone tissue repair.
Guizzardi S, Galli C, Govoni P, Boratto R, Cattarini G, Martini D, Belletti S, Scandroglio R.
Source
Department of Experimental Medicine–Section of Histology, Via Volturno 39, University of Parma, 43100 Parma, Italy. stegui@unipr.it
Abstract
Several researchers have recently shed new light upon the importance of extracellular nucleotides and nucleosides to stimulate cells growth.
PDRN, a mixture of deoxyribonucleotides polymers of different lengths, has recently demonstrated to stimulate “in vitro” fibroblast proliferation and collagen production, probably stimulating the purinergic receptor system.
In this work we evaluated the effects of PDRN on human cultured osteoblasts, focusing our attention on cell proliferation and alkaline phosphatase activity. PDRN at a concentration of 100 microg/ml induce an increase in osteoblasts growth after 6 days as compared to control (+21%). The addition of DMPX 50 microM and suramine (P2 inhibitor) 10 microM give different results: suramine has no significant effect, while DPMX reduce, even if partially, the PDRN induced cell growth. The alkaline phosphatase activity shows a gradual enhancement starting from day 0 to day 10, even if PDRN treated cells, examined at day 6, present a sensibly lower phosphatase activity when compared to controls.
Our data demonstrate that PDRN acts as an osteoblast growth stimulator.
Its action is partially due to a stimulation of the purinergic system mediated by A2 purinoreceptors, however we can not exclude the involvement of other mechanism like salvage pathway.
(PDRN = mixture of Nucleotides)
PMID:12899922[PubMed – indexed for MEDLINE]

Crit Care Med. 2008 May;36(5):1594-602.
Polydeoxyribonucleotide improves angiogenesis and wound healing in experimental thermal injury.
Bitto A, Galeano M, Squadrito F, Minutoli L, Polito F, Dye JF, Clayton EA, Calò M, Venuti FS, Vaccaro M, Altavilla D.
Source   Department of Clinical and Experimental Medicine and Pharmacology, Section of Pharmacology, University of Messina, Italy.
Abstract
Polydeoxyribonucleotide contains a mixture of nucleotides and interacts with adenosine receptors, stimulating vascular endothelial growth factor expression and wound healing.
The purpose of this study was to investigate the effect of polydeoxyribonucleotide on experimental burn wounds.
DESIGN:    Randomized experiment.
SETTING:    Research laboratory at a university hospital.
SUBJECTS:   Thermal injury in mice.
INTERVENTIONS:
Mice were immersed in 80 degrees C water for 10 secs to achieve a deep-dermal second-degree burn. Animals were randomized to receive either polydeoxyribonucleotide (8 mg/kg/day intraperitoneally for 14 days) or its vehicle alone (0.9% NaCl solution at 100 microL/day intraperitoneally). On days 7 and 14 the animals were killed. Blood was collected for tumor necrosis factor-alpha measurement; burn areas were used for histologic and immunohistochemical examination, for the evaluation of vascular endothelial growth factor and nitric oxide synthases by Western blot, and for the determination of wound nitric oxide products.
MEASUREMENTS AND MAIN RESULTS:
Polydeoxyribonucleotide increased burn wound re-epithelialization and reduced the time to final wound closure. Polydeoxyribonucleotide improved healing of burn wound through increased epithelial proliferation and maturation of the extracellular matrix as confirmed by fibronectin and laminin immunostaining. Polydeoxyribonucleotide also improved neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of platelet-endothelial cell adhesion molecule-1. Furthermore, polydeoxyribonucleotide blunted serum tumor necrosis factor-alpha and enhanced inducible nitric oxide synthase and vascular endothelial growth factor expression and the wound content of nitric oxide products.
CONCLUSIONS:
Our study suggests that polydeoxyribonucleotide may be an effective therapeutic approach to improve clinical outcomes after thermal injury.
(PDRN =  mixture of Nucleotides)
PMID:18434887[PubMed – indexed for MEDLINE]

J Cell Physiol. 2012 Jun;227(6):2622-31.
CD73-generated adenosine promotes osteoblast differentiation.
Takedachi M, Oohara H, Smith BJ, Iyama M, Kobashi M, Maeda K, Long CL, Humphrey MB, Stoecker BJ, Toyosawa S, Thompson LF, Murakami S.
Source
Department of Periodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.
Abstract
CD731 is a GPI-anchored cell surface protein with ecto-5′-nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73-generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73-deficient (cd73(-/-)) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild-type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3-E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3-E1 cells expressed adenosine A(2A) receptors (A(2A)AR) and A(2B) receptors (A(2B)AR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3-E1 cells over expressing CD73 were suppressed by treatment with an A(2B)AR antagonist but not with an A(2A) AR antagonist.
Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A(2B)AR signaling.
Copyright © 2011 Wiley Periodicals, Inc PMID:20331607[PubMed – indexed for MEDLINE] PMCID:PMC2860211

Am J Pathol. 2012 Feb;180(2):775-86.
Adenosine A(2A) receptor ligation inhibits osteoclast formation.
Mediero A, Kara FM, Wilder T, Cronstein BN.
Source
Division of Translational Medicine, Department of Medicine, New York University School of Medicine, New York, NY 10016, USA.
Abstract
Adenosine is generated in increased concentrations at sites of injury/hypoxia and mediates a variety of physiological and pharmacological effects via G protein-coupled receptors (A(1), A(2A), A(2B), and A(3)).
Because all adenosine receptors are expressed on osteoclasts, we determined the role of A(2A) receptor in the regulation of osteoclast differentiation.
Differentiation and bone resorption were studied as the macrophage colony-stimulating factor-1-receptor activator of NF-κB ligand formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells from primary murine bone marrow-derived precursors. A(2A) receptor and osteoclast marker expression levels were studied by RT-PCR. Cytokine secretion was assayed by enzyme-linked immunosorbent assay. In vivo examination of A(2A) knockout (KO)/control bones was determined by TRAP staining, micro-computed tomography, and electron microscopy. The A(2A) receptor agonist, CGS21680, inhibited osteoclast differentiation and function (half maximal inhibitory concentration, 50 nmol/L), increased the percentage of immature osteoclast precursors, and decreased IL-1β and tumor necrosis factor-α secretion, an effect that was reversed by the A(2A) antagonist, ZM241385. Cathepsin K and osteopontin mRNA expression increased in control and ZM241385-pretreated osteoclasts, and this was blocked by CGS21680. Micro-computed tomography of A(2A)KO mouse femurs showed a significantly decreased bone volume/trabecular bone volume ratio, decreased trabecular number, and increased trabecular space. A(2A)KO femurs showed an increased TRAP-positive osteoclast. Electron microscopy in A(2A)KO femurs showed marked osteoclast membrane folding and increased bone resorption.
Thus, adenosine, acting via the A(2A) receptor, inhibits macrophage colony-stimulating factor-1-receptor activator of NF-κB ligand-stimulated osteoclast differentiation and may regulate bone turnover under conditions in which adenosine levels are elevated.
Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
PMID:22138579[PubMed – indexed for MEDLINE] PMCID:PMC3349861

Exp Cell Res. 2013 Aug 1;319(13):2028-36.
ATP and UTP stimulate bone morphogenetic protein-2,-4 and -5 gene expression and mineralization by rat primary osteoblasts involving PI3K/AKT pathway.
Ayala-Peña VB, Scolaro LA, Santillán GE.
Source
Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, Bahía Blanca, Buenos Aires B8000ICN, Argentina.
Abstract
The modulation of purinergic receptors plays an important role in the regulation of bone formation by the osteoblast.
On the other hand, bone morphogenetic proteins (BMPs), members of the transforming growth factor-β superfamily, regulate the differentiation of osteoprogenitor bone cells and stimulate bone formation.
In this study, we investigate the effects of several nucleotides on osteoblast differentiation and function, and their relation with the gene expression of osteogenic proteins BMP-2, BMP-4 and BMP-5 as well as of differentiation markers alkaline phosphatase (ALP) and bone sialoprotein (BSP). Our results indicate that 100μM ATP, ATPγS and UTP, but not ADP, ADPβS or UDP, promote ALP activity in rat primary osteoblasts, showing a peak about day 7 of the treatment. ATP, ATPγS and UTP also increase the mRNA levels of ALP, BMP-2, BMP-4, BMP-5 and BSP. Both the ALP activity and ALP and BMP-4 mRNA increments induced by ATP and UTP are inhibited by Ly294002, a PI3K inhibitor, suggesting the involvement of PI3K/AKT signaling pathway in purinergic modulation of osteoblast differentiation. Furthermore, bone mineralization enhance 1 and 1.5 fold after culturing osteoblasts in the presence of 100μM ATP or UTP, respectively, but not of ADP or UDP for 22 days. This information suggests that P2Y2 receptors (responsive to ATP, ATPγS and UTP) enhance osteoblast differentiation involving PI3K/AKT signaling pathway activation and gene expression induction of ALP, BMP-2, BMP-4, BMP-5 and BSP. Our findings state a novel molecular mechanism that involves specific gene expression activation of osteoblast function by the purinoreceptors, which would be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.
Copyright © 2013 Elsevier Inc. All rights reserved.PMID:23707969[PubMed – in process]