International Union of Basic and Clinical Pharmacology. CXII: Adenosine Receptors: A Further Update.

Our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors (2011) contained a number of emerging developments with respect to this G protein-coupled receptor subfamily, including protein structure, protein oligomerization, protein diversity, and allosteric modulation by small molecules. Since then, a wealth of new data and results has been added, allowing us to explore novel concepts such as target binding kinetics and biased signaling of adenosine receptors, to examine a multitude of receptor structures and novel ligands, to gauge new pharmacology, and to evaluate clinical trials with adenosine receptor ligands. This review should therefore be considered a further update of our previous reports from 2001 and 2011. SIGNIFICANCE STATEMENT: Adenosine receptors (ARs) are of continuing interest for future treatment of chronic and acute disease conditions, including inflammatory diseases, neurodegenerative afflictions, and cancer. The design of AR agonists ("biased" or not) and antagonists is largely structure based now, thanks to the tremendous progress in AR structural biology. The A2A- and A2BAR appear to modulate the immune response in tumor biology. Many clinical trials for this indication are ongoing, whereas an A2AAR antagonist (istradefylline) has been approved as an anti-Parkinson agent.

Adenosine A2A receptor activation reduces chondrocyte senescence.

Osteoarthritis (OA) pathogenesis is associated with reduced chondrocyte homeostasis and increased levels of cartilage cellular senescence. Chondrosenescence is the development of cartilage senescence that increases with aging joints and disrupts chondrocyte homeostasis and is associated with OA. Adenosine A2A receptor (A2AR) activation in cartilage via intra-articular injection of liposomal A2AR agonist, liposomal-CGS21680, leads to cartilage regeneration in vivo and chondrocyte homeostasis. A2AR knockout mice develop early OA isolated chondrocytes demonstrate upregulated expression of cellular senescence and aging-associated genes. Based on these observations, we hypothesized that A2AR activation would ameliorate cartilage senescence. We found that A2AR stimulation of chondrocytes reduced beta-galactosidase staining and regulated levels and cell localization of common senescence mediators p21 and p16 in vitro in the human TC28a2 chondrocyte cell line. In vivo analysis similarly showed A2AR activation reduced nuclear p21 and p16 in obesity-induced OA mice injected with liposomal-CGS21680 and increased nuclear p21 and p16 in A2AR knockout mouse chondrocytes compared to wild-type mice. A2AR agonism also increased activity of the chondrocyte Sirt1/AMPK energy-sensing pathway by enhancing nuclear Sirt1 localization and upregulating T172-phosphorylated (active) AMPK protein levels. Lastly, A2AR activation in TC28a2 and primary human chondrocytes reduced wild-type p53 and concomitantly increased p53 alternative splicing leading to increase in an anti-senescent p53 variant, Δ133p53α. The results reported here indicate that A2AR signaling promotes chondrocyte homeostasis in vitro and reduces OA cartilage development in vivo by reducing chondrocyte senescence.

Adenosine A2A receptor (A2AR) stimulation enhances mitochondrial metabolism and mitigates reactive oxygen species-mediated mitochondrial injury.

In OA chondrocytes, there is diminished mitochondrial production of ATP and diminished extracellular adenosine resulting in diminished adenosine A2A receptor (A2AR) stimulation and altered chondrocyte homeostasis which contributes to the pathogenesis of OA. We tested the hypothesis that A2AR stimulation maintains or enhances mitochondrial function in chondrocytes. The effect of A2AR signaling on mitochondrial health and function was determined in primary murine chondrocytes, a human chondrocytic cell line (T/C-28a2), primary human chondrocytes, and a murine model of OA by transmission electron microscopy analysis, mitochondrial stress testing, confocal live imaging for mitochondrial inner membrane polarity, and immunohistochemistry. In primary murine chondrocytes from A2AR-/- null mice, which develop spontaneous OA by 16 weeks, there is mitochondrial swelling, dysfunction, and reduced mitochondrial content with increased reactive oxygen species (ROS) burden and diminished mitophagy, as compared to chondrocytes from WT animals. IL-1-stimulated T/C-28a2 cells treated with an A2AR agonist had reduced ROS burden with increased mitochondrial dynamic stability and function, findings which were recapitulated in primary human chondrocytes. In an obesity-induced OA mouse model, there was a marked increase in mitochondrial oxidized material which was markedly improved after intraarticular injections of liposomal A2AR agonist. These results are consistent with the hypothesis that A2AR ligation is mitoprotective in OA.

Adenosine A2A receptor null chondrocyte transcriptome resembles that of human osteoarthritic chondrocytes.

Adenosine signaling plays a critical role in the maintenance of articular cartilage and may serve as a novel therapeutic for osteoarthritis (OA), a highly prevalent and morbid disease without effective therapeutics in the current market. Mice lacking adenosine A2A receptors (A2AR) develop spontaneous OA by 16 weeks of age, a finding relevant to human OA since loss of adenosine signaling due to diminished adenosine production (NT5E deficiency) also leads to development of OA in mice and humans. To better understand the mechanism by which A2AR and adenosine generation protect from OA development, we examined differential gene expression in neonatal chondrocytes from WT and A2AR null mice. Analysis of differentially expressed genes was analyzed by KEGG pathway analysis, and oPOSSUM and the flatiron database were used to identify transcription factor binding enrichment, and tissue-specific network analyses and patterns were compared to gene expression patterns in chondrocytes from patients with OA. There was a differential expression of 2211 genes (padj<0.05). Pathway enrichment analysis revealed that pro-inflammatory changes, increased metalloprotease, reduced matrix organization, and homeostasis are upregulated in A2AR null chondrocytes. Moreover, stress responses, including autophagy and HIF-1 signaling, seem to be important drivers of OA and bear marked resemblance to the human OA transcriptome. Although A2AR null mice are born with grossly intact articular cartilage, we identify here the molecular foundations for early-onset OA in these mice, further establishing their role as models for human disease and the potential use of adenosine as a treatment for human disease.

Transforming the Degradation Rate of ß-tricalcium Phosphate Bone Replacement Using 3-Dimensional Printing.

BACKGROUND: ß-Tricalcium phosphate (ß-TCP) is one of the most common synthetic bone grafting materials utilized in craniofacial reconstruction; however, it is limited by a slow degradation rate. The aim of this study was to leverage 3-dimensional (3D) printing in an effort to accelerate the degradation kinetics of ß-TCP. METHODS: Twenty-two 1-month-old New Zealand white rabbits underwent creation of calvarial and alveolar defects, repaired with 3D-printed ß-TCP scaffolds coated with 1000 µM of osteogenic agent dipyridamole. Rabbits were euthanized after 2, 6, and 18 months after surgical intervention. Bone regeneration, scaffold degradation, and bone mechanical properties were quantified. RESULTS: Histological analysis confirmed the generation of vascularized and organized bone. Microcomputed tomography analysis from 2 to 18 months demonstrated decreased scaffold volume within calvarial (23.6% ± 2.5%, 5.1% ± 2.2%; P < 0.001) and alveolar (21.5% ± 2.2%, 0.2% ± 1.9%; P < 0.001) defects, with degradation rates of 54.6%/year and 90.5%/year, respectively. Scaffold-inducted bone generation within the defect was volumetrically similar to native bone in the calvarium (55.7% ± 6.9% vs 46.7% ± 6.8%; P = 0.064) and alveolus (31.4% ± 7.1% vs 33.8% ± 3.7%; P = 0.337). Mechanical properties between regenerated and native bone were similar. CONCLUSIONS: Our study demonstrates an improved degradation profile and replacement of absorbed ß-TCP with vascularized, organized bone through 3D printing and addition of an osteogenic agent. This novel additive manufacturing and tissue engineering protocol has implications to the future of craniofacial skeletal reconstruction as a safe and efficacious bone tissue engineering method.

Unmet need in rheumatology: reports from the Targeted Therapies meeting 2019.

OBJECTIVES: To detail the greatest areas of unmet scientific and clinical needs in rheumatology. METHODS: The 21st annual international Advances in Targeted Therapies meeting brought together more than 100 leading basic scientists and clinical researchers in rheumatology, immunology, epidemiology, molecular biology and other specialties. During the meeting, breakout sessions were convened, consisting of 5 disease-specific groups with 20-30 experts assigned to each group based on expertise. Specific groups included: rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, systemic lupus erythematosus and other systemic autoimmune rheumatic diseases. In each group, experts were asked to identify unmet clinical and translational research needs in general and then to prioritise and detail the most important specific needs within each disease area. RESULTS: Overarching themes across all disease states included the need to innovate clinical trial design with emphasis on studying patients with refractory disease, the development of trials that take into account disease endotypes and patients with overlapping inflammatory diseases, the need to better understand the prevalence and incidence of inflammatory diseases in developing regions of the world and ultimately to develop therapies that can cure inflammatory autoimmune diseases. CONCLUSIONS: Unmet needs for new therapies and trial designs, particularly for those with treatment refractory disease, remain a top priority in rheumatology.

Adenosine A2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of ß-catenin in osteoblasts.

Osteoblast differentiation and proliferation are regulated by several modulators, among which are adenosine A2A receptors (A2ARs) and Wingless/Integrated-ß-catenin pathways. Cytosolic ß-catenin stabilization promotes its nuclear translocation and transcriptional activity. In the present study, we seek to determine whether there is a connection between A2AR stimulation and cellular ß-catenin levels in osteoblasts. Osteoblast precursor cell line (MC3T3-E1) and primary murine osteoblasts were treated with CGS21680, a highly selective A2AR agonist. We analyzed cellular content and nuclear translocation of phosphorylated (p)-serine 552 (S552) ß-catenin in response to A2AR stimulation in MC3T3-E1 cells, in both wild-type and A2AR knockout (A2AKO) mice. Moreover, we measured cellular ß-catenin levels in MC3T3-E1 cells transfected with scrambled or protein kinase B (Akt) small interfering RNA following A2AR activation. CGS21680 (1 µM) stimulated an increase in both the cellular content and nuclear translocation of p-S552 ß-catenin after 15 min of incubation. A2AR activation had no tangible effect on the cellular ß-catenin level either in A2AKO mice or in osteoblasts with diminished Akt content. Our findings demonstrate an interaction between A2AR, ß-catenin, and Akt signaling in osteoblasts. The existence of such a crosstalk has significant repercussions in the development of novel therapeutic approaches targeting medical conditions associated with reduced bone density.-Borhani, S., Corciulo, C., Larranaga-Vera, A., Cronstein, B. N. Adenosine A2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of ß-catenin in osteoblasts.

Unmet need in rheumatology: reports from the Targeted Therapies meeting 2018.

To develop a comprehensive listing of the greatest unmet scientific and clinical needs in rheumatology. The 20th annual international Targeted Therapies meeting brought more than 100 leading basic scientists and clinical researchers in rheumatology, immunology, epidemiology, molecular biology and other specialties. During the meeting, breakout sessions were convened, consisting of five disease-specific groups with 20-30 experts assigned to each group based on expertise. Specific groups included rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, systemic lupus erythematosus, connective tissue diseases and a basic science immunology group spanning all of these clinical domains. In each group, experts were asked to consider recent accomplishments within their clinical domain in the last year and update the unmet needs in three categorical areas: basic/translational science, clinical science and therapeutic development, and clinical care. While progress was noted among some of previously identified needs, both new needs were identified and themes from prior meetings were re-iterated: the need for better understanding the heterogeneity within each disease, and for identifying preclinical states of disease allowing treatment and prevention of disease in those at risk, and the elusive ability to cure disease. Within the clinical care realm, improved comorbidity management and patient-centred care continue to be unmet needs, and the need for new and affordable therapeutics was highlighted. Unmet needs for new and accessible targeted therapies, disease prevention and ultimately cure remain a priority in rheumatology.

Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.

The axonal guidance proteins semaphorin (Sema)4D and Sema3A play important roles in communication between osteoclasts and osteoblasts. As stimulation of adenosine A2A receptors (A2AR) regulates both osteoclast and osteoblast function, we asked whether A2AR regulates both osteoclast and osteoblast expression of Semas. In vivo bone formation and Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1 protein expression were studied in a murine model of wear particle-induced osteolysis. Osteoclast/osteoblast differentiation were studied in vitro as the number of tartrate-resistant acid phosphatase+/Alizarin Red+ cells after challenge with CGS21680 (A2AR agonist, 1 µM) or ZM241385 (A2AR antagonist, 1 µM), with or without Sema4D or Sema3A (10 ng/ml). Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1, and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) expression was studied by RT-PCR and Western blot. ß-Catenin activation and cytoskeleton changes were studied by fluorescence microscopy and Western blot. In mice with wear particles implanted over the calvaria, CGS21680 treatment increased bone formation in vivo, reduced Sema4D, and increased Sema3A expression compared with mice with wear particle-induced osteolysis treated with vehicle alone. During osteoclast differentiation, CGS21680 abrogated RANKL-induced Sema4D mRNA expression (1.3 ± 0.3- vs. 2.5 ± 0.1-fold change, P < 0.001, n = 4). PlexinA1, but not Neuropilin-1, mRNA was enhanced by CGS21680 treatment. CGS21680 enhanced Sema3A mRNA expression during osteoblast differentiation (8.7 ± 0.2-fold increase, P < 0.001, n = 4); PlexinB1 mRNA was increased 2-fold during osteoblast differentiation and was not altered by CGS21680. Similar changes were observed at the protein level. CGS21680 decreased RANKL, increased OPG, and increased total/nuclear ß-catenin expression in osteoblasts. Sema4D increased Ras homolog gene family, member A phosphorylation and focal adhesion kinase activation in osteoclast precursors, and CGS21680 abrogated these effects. In summary, A2AR activation diminishes secretion of Sema4D by osteoclasts, inhibits Sema4D-mediated osteoclast activation, and enhances secretion of Sema3A by osteoblasts, increasing osteoblast differentiation and diminishing inflammatory osteolysis.-Mediero, A., Wilder, T., Shah, L., Cronstein, B. N. Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.

Three dimensionally printed bioactive ceramic scaffold osseoconduction across critical-sized mandibular defects.

BACKGROUND: Vascularized bone tissue transfer, commonly used to reconstruct large mandibular defects, is challenged by long operative times, extended hospital stay, donor-site morbidity, and resulting health care. 3D-printed osseoconductive tissue-engineered scaffolds may provide an alternative solution for reconstruction of significant mandibular defects. This pilot study presents a novel 3D-printed bioactive ceramic scaffold with osseoconductive properties to treat segmental mandibular defects in a rabbit model. METHODS: Full-thickness mandibulectomy defects (12 mm) were created at the mandibular body of eight adult rabbits and replaced by 3D-printed ceramic scaffold made of 100% ß-tricalcium phosphate, fit to defect based on computed tomography imaging. After 8 weeks, animals were euthanized, the mandibles were retrieved, and bone regeneration was assessed. Bone growth was qualitatively assessed with histology and backscatter scanning electron microscopy, quantified both histologically and with micro computed tomography and advanced 3D image reconstruction software, and compared to unoperated mandible sections (UMSs). RESULTS: Histology quantified scaffold with newly formed bone area occupancy at 54.3 ± 11.7%, compared to UMS baseline bone area occupancy at 55.8 ± 4.4%, and bone area occupancy as a function of scaffold free space at 52.8 ± 13.9%. 3D volume occupancy quantified newly formed bone volume occupancy was 36.3 ± 5.9%, compared to UMS baseline bone volume occupancy at 33.4 ± 3.8%, and bone volume occupancy as a function of scaffold free space at 38.0 ± 15.4%. CONCLUSIONS: 3D-printed bioactive ceramic scaffolds can restore critical mandibular segmental defects to levels similar to native bone after 8 weeks in an adult rabbit, critical sized, mandibular defect model.

Adenosine A2A receptor plays an important role in radiation-induced dermal injury.

Ionizing radiation is a common therapeutic modality and following irradiation dermal changes, including fibrosis and atrophy, may lead to permanent changes. We have previously demonstrated that occupancy of A2A receptor (A2AR) stimulates collagen production, so we determined whether blockade or deletion of A2AR could prevent radiation-induced fibrosis. After targeted irradiation (40 Gy) of the skin of wild-type (WT) or A2AR knockout (A2ARKO) mice, the A2AR antagonist ZM241385 was applied daily for 28 d. In irradiated WT mice treated with the A2AR antagonist, there was a marked reduction in collagen content and skin thickness, and ZM241385 treatment reduced the number of myofibroblasts and angiogenesis. After irradiation, there is an increase in loosely packed collagen fibrils, which is significantly diminished by ZM241385. Irradiation also induced an increase in epidermal thickness, prevented by ZM241385, by increasing the number of proliferating keratinocytes. Similarly, in A2ARKO mice, the changes in collagen alignment, skin thickness, myofibroblast content, angiogenesis, and epidermal hyperplasia were markedly reduced following irradiation. Radiation-induced changes in the dermis and epidermis were accompanied by an infiltrate of T cells, which was prevented in both ZM241385-treated and A2ARKO mice. Radiation therapy is administered to a significant number of patients with cancer, and radiation reactions may limit this therapeutic modality. Our findings suggest that topical application of an A2AR antagonist prevents radiation dermatitis and may be useful in the prevention or amelioration of radiation changes in the skin.

Netrin-1 and its receptor Unc5b are novel targets for the treatment of inflammatory arthritis.

Rheumatoid arthritis is an autoimmune disease that is characterized by chronic inflammation and destruction of joints. Netrin-1, a chemorepulsant, laminin-like matrix protein, promotes inflammation by preventing macrophage egress from inflamed sites and is required for osteoclast differentiation. We asked whether blockade of Netrin-1 or its receptors [Unc5b and DCC (deleted in colorectal carcinoma)] may be useful therapeutic targets for treatment of inflammatory arthritis. Arthritis was induced in 8-wk-old C57Bl/6 mice by intraperitoneal injection of K/BxN serum. Murine monoclonal antibodies against Netrin-1, Unc5b, or DCC (10 µg/mouse) were injected weekly for 4 wk (n = 10). Paw swelling and thickness were assessed and following euthanasia 2-4 wk after serum transfer, paws were prepared for micro-computed tomography and histology. Paw inflammation was maximal 2 wk after injection. Anti-Netrin-1 or anti-Unc5b, but not anti-DCC, antibodies significantly reduced paw inflammation (clinical score: 9.8 ± 0.8, 10.4 ± 0.9, and 13.5 ± 0.5, respectively vs 16 ± 0 for control; P < 0.001). Micro-computed tomography showed bony erosions in untreated or anti-DCC-treated mice, whereas there were no erosions in anti-Netrin-1/anti-Unc5b-treated-animals. Tartrate-resistant acid phosphatase staining demonstrated a marked decrease in osteoclasts in anti-Netrin-1/anti-Unc5b-treated animals. Immunofluorescence staining revealed a decrease in cathepsin K+ and CD68+ cells in anti-Netrin-1/anti-Unc5b-treated animals. Blockade of Netrin-1/Unc5b by monoclonal antibodies prevents bone destruction and reduces the severity of K/BxN serum transfer-induced arthritis. Netrin-1 may be a novel therapeutic target for treatment of inflammatory bone destruction.-Mediero, A., Wilder, T., Ramkhelawon, B., Moore, K. J., Cronstein, B. N. Netrin-1 and its receptor Unc5b are novel targets for the treatment of inflammatory arthritis.

Ticagrelor regulates osteoblast and osteoclast function and promotes bone formation in vivo via an adenosine-dependent mechanism.

As many as 10% of bone fractures heal poorly, and large bone defects resulting from trauma, tumor, or infection may not heal without surgical intervention. Activation of adenosine A2A receptors (A2ARs) stimulates bone formation. Ticagrelor and dipyridamole inhibit platelet function by inhibiting P2Y12 receptors and platelet phosphodiesterase, respectively, but share the capacity to inhibit cellular uptake of adenosine and thereby increase extracellular adenosine levels. Because dipyridamole promotes bone regeneration by an A2AR-mediated mechanism we determined whether ticagrelor could regulate the cells involved in bone homeostasis and regeneration in a murine model and whether inhibition of P2Y12 or indirect A2AR activation via adenosine was involved. Ticagrelor, dipyridamole and the active metabolite of clopidogrel (CAM), an alternative P2Y12 antagonist, inhibited osteoclast differentiation and promoted osteoblast differentiation in vitro. A2AR blockade abrogated the effects of ticagrelor and dipyridamole on osteoclast and osteoblast differentiation whereas A2BR blockade abrogated the effects of CAM. Ticagrelor and CAM, when applied to a 3-dimentional printed resorbable calcium-triphosphate/hydroxyapatite scaffold implanted in a calvarial bone defect, promoted significantly more bone regeneration than the scaffold alone and as much bone regeneration as BMP-2, a growth factor currently used to promote bone regeneration. These results suggest novel approaches to targeting adenosine receptors in the promotion of bone regeneration.-Mediero, A., Wilder, T., Reddy, V. S. R., Cheng, Q., Tovar, N., Coelho, P. G., Witek, L., Whatling, C., Cronstein, B. N. Ticagrelor regulates osteoblast and osteoclast function and promotes bone formation in vivo via an adenosine-dependent mechanism.

Netrin-1 is highly expressed and required in inflammatory infiltrates in wear particle-induced osteolysis.

OBJECTIVE: Netrin-1 is a chemorepulsant and matrix protein expressed during and required for osteoclast differentiation, which also plays a role in inflammation by preventing macrophage egress. Because wear particle-induced osteolysis requires osteoclast-mediated destruction of bone, we hypothesised that blockade of Netrin-1 or Unc5b, a receptor for Netrin-1, may diminish this pathological condition. METHODS: C57BL/6 mice, 6-8 weeks old, had 3 mg of ultrahigh-molecular-weight polyethylene particles implanted over the calvaria and then received 10 µg of monoclonal antibodies for Netrin-1 or its receptors, Unc5b and deleted in colon cancer (DCC), injected intraperitoneally on a weekly basis. After 2 weeks, micro-computed tomography and histology analysis were performed. Netrin-1 expression was analysed in human tissue obtained following primary prosthesis implantation or after prosthesis revision for peri-implant osteolysis and aseptic implant loosening. RESULTS: Weekly injection of anti-Netrin-1 or anti-Unc5b-antibodies significantly reduced particle-induced bone pitting in calvaria exposed to wear particles (46±4% and 49±3% of control bone pitting, respectively, p<0.001) but anti-DCC antibody did not affect inflammatory osteolysis (80±7% of control bone pitting, p=ns). Anti-Netrin-1 or anti-Unc5b, but not anti-DCC, antibody treatment markedly reduced the inflammatory infiltrate and the number of tartrate resistance acid phosphatase (TRAP)-positive osteoclasts (7±1, 4±1 and 14±1 cells/high power field (hpf), respectively, vs 12±1 cells/hpf for control, p<0.001), with no significant changes in alkaline phosphatase-positive osteoblasts on bone-forming surfaces in any antibody-treated group. Netrin-1 immunostaining colocalised with CD68 staining for macrophages. The peri-implant tissues of patients undergoing prosthesis revision surgery showed an increase in Netrin-1 expression, whereas there was little Netrin-1 expression in soft tissues removed at the time of primary joint replacement. CONCLUSIONS: These results demonstrate a unique role for Netrin-1 in osteoclast biology and inflammation and may be a novel target for prevention/treatment of inflammatory osteolysis.

Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2.

Promoting bone regeneration and repair of bone defects is a need that has not been well met to date. We have previously found that adenosine, acting via A2A receptors (A2AR) promotes wound healing and inhibits inflammatory osteolysis and hypothesized that A2AR might be a novel target to promote bone regeneration. Therefore, we determined whether direct A2AR stimulation or increasing endogenous adenosine concentrations via purine transport blockade with dipyridamole regulates bone formation. We determined whether coverage of a 3 mm trephine defect in a mouse skull with a collagen scaffold soaked in saline, bone morphogenetic protein-2 (BMP-2; 200 ng), 1 µM CGS21680 (A2AR agonist, EC50 = 160 nM), or 1 µM dipyridamole (EC50 = 32 nM) promoted bone regeneration. Microcomputed tomography examination demonstrated that CGS21680 and dipyridamole markedly enhanced bone regeneration as well as BMP-2 8 wk after surgery (60 ± 2%, 79 ± 2%, and 75 ± 1% bone regeneration, respectively, vs. 32 ± 2% in control, P < 0.001). Blockade by a selective A2AR antagonist (ZM241385, 1 µM) or deletion of A2AR abrogated the effect of CGS21680 and dipyridamole on bone regeneration. Both CGS21680 and dipyridamole treatment increased alkaline phosphatase-positive osteoblasts and diminished tartrate resistance acid phosphatase-positive osteoclasts in the defects. In vivo imaging with a fluorescent dye for new bone formation revealed a strong fluorescent signal in treated animals that was equivalent to BMP-2. In conclusion, stimulation of A2AR by specific agonists or by increasing endogenous adenosine levels stimulates new bone formation as well as BMP-2 and represents a novel approach to stimulating bone regeneration.

Regulation of bone and cartilage by adenosine signaling.

There is growing recognition that bone serves important endocrine and immunologic functions that are compromised in several disease states. While many factors are known to affect bone metabolism, recent attention has focused on investigating the role of purinergic signaling in bone formation and regulation. Adenosine is a purine nucleoside produced intracellularly and extracellularly in response to stimuli such as hypoxia and inflammation, which then interacts with P1 receptors. Numerous studies have suggested that these receptors play a pivotal role in osteoblast, osteoclast, and chondrocyte differentiation and function. This review discusses the various ways by which adenosine signaling contributes to bone and cartilage homeostasis, while incorporating potential therapeutic applications of these signaling pathways.

Adenosine A2B receptors play an important role in bone homeostasis.

Bone homeostasis is a finely regulated mechanism involving different molecular pathways including adenosine signaling. The aim of this study is to determine the bone phenotype of adenosine A2B receptor knockout (A2BRKO) mice and to measure their ability to form new bone. Moreover, we analyzed the functionality of osteoclasts and osteoblasts from A2BRKO mice. Microcomputed tomography (µCT) analysis revealed a decrease of bone substance, bone mineral density, and trabecular number in A2BRKO mice compared to the WT mice at the same age. We measured the new bone formation by injecting fluorescent markers: it was reduced in femur and tibia of A2BRKO mice compare to the WT. A2BRKO young mice have fewer osteoblasts and an increase of osteoclasts was measured in the hind limbs of young and adult mice. A2BRKO osteoclasts are also more active in vitro, showing an increase of pit formation in dentin discs. Surprisingly in mature osteoblasts from A2BRKO mice, we measured an increase of calcified matrix production, collagen deposition, and alkaline phosphatase activity. These results demonstrate that A2BR on osteoblasts and osteoclasts regulate bone homeostasis.

Activation of EPAC1/2 is essential for osteoclast formation by modulating NFκB nuclear translocation and actin cytoskeleton rearrangements.

Bisphosphonates inhibit osteoclast differentiation/function via inhibition of Rap1A isoprenylation. As Rap1 is the effector of exchange protein directly activated by cAMP (EPAC) proteins, we determined the role of EPAC in osteoclast differentiation. We examined osteoclast differentiation as the number of primary murine/human bone-marrow precursors that differentiated into multinucleated TRAP-positive cells in the presence of EPAC-selective stimulus (8-pCTP-2'-O-Me-cAMP, 100 µM; 8-pCTP-2'-O-Me-cAMP-AM, 1 µM) or inhibitor brefeldin A (BFA), ESI-05, and ESI-09 (10 µM each). Rap1 activity was assessed, and signaling events, as well as differentiation in EPAC1/2-knockdown RAW264.7 cells, were studied. Direct EPAC1/2 stimulation significantly increased osteoclast differentiation, whereas EPAC1/2 inhibition diminished differentiation (113 ± 6%, P < 0.05, and 42 ± 10%, P < 0.001, of basal, respectively). Rap1 activation was maximal 15 min after RANKL stimulation (147 ± 9% of basal, P < 0.001), whereas silencing of EPAC1/2 diminished activated Rap1 (43 ± 13 and 20 ± 15% of control, P < 0.001) and NFkB nuclear translocation. TRAP-staining revealed no osteoclast differentiation in EPAC1/2-KO cells. Cathepsin K, NFATc1, and osteopontin mRNA expression decreased in EPAC1/2-KO cells when compared to control. RhoA, cdc42, Rac1, and FAK were activated in an EPAC1/2-dependent manner, and there was diminished cytoskeletal assembly in EPAC1/2-KO cells. In summary, EPAC1 and EPAC2 are critical signaling intermediates in osteoclast differentiation that permit RANKL-stimulated NFkB nuclear translocation and actin rearrangements. Targeting this signaling intermediate may diminish bone destruction in inflammatory arthritis.