Extracellular adenosine signaling in bone health and disease

Abstract

Purinergic signaling is a key molecular pathway in the maintenance of bone health and regeneration. P1 receptor signaling, which is activated by extracellular adenosine, has emerged as a key metabolic pathway that regulates bone tissue formation, function, and homeostasis. Extracellular adenosine is mainly produced by ectonucleotidases, and alterations in the function of these enzymes or compromised adenosine generation can result in bone disorders, such as osteoporosis and impaired fracture healing. This mini review discusses the key role played by adenosine in bone health and how its alterations contribute to bone diseases, as well as potential therapeutic applications of exogenous adenosine to combat bone diseases like osteoporosis and injury.

Introduction

Bone is a dynamic tissue which continuously undergoes remodeling primarily through osteoblast-mediated bone formation and osteoclast-mediated bone resorption [1]. The close interplay between osteoblast and osteoclast activity ultimately determines the turnover, remodeling, and overall health of bone tissue. Disruption of this homeostasis due to alterations in molecular signaling, physiological aging, or injury can lead to various bone pathologies and compromised healing. Purinergic signaling has been identified as a key molecular pathway involved in bone remodeling and homeostasis [2]. Purinergic receptors can be categorized into either P2- or P1-type based on the activation ligand. P2 receptors are activated by nucleotides whereas P1 receptors are activated by the nucleoside adenosine. While P2 and P1 purinergic receptors are expressed by osteoblasts and osteoclasts, this review will focus on the role played by purinergic P1 receptors and adenosine signaling in bone tissue health and regeneration. For a more comprehensive review on P2 purinergic signaling and its effect on bone homeostasis, refer to other reviews [3].

Adenosine is a nucleoside that is produced mostly through enzymatic degradation of adenine nucleotides (ATP, ADP, and AMP). Typically, healthy physiological concentrations of adenosine are maintained between 20 and 300 nM [4]. Extracellular adenosine levels can be drastically increased in response to pathological conditions such as injury, inflammation, or factors that contribute to cellular stresses; where cells can produce over 10 times the basal level of adenosine, resulting in micromolar concentrations [5]. This effect is a result of a stress-induced increase in phosphatase activity along with activation of ecto-5’-nucleotidase (CD73) and ectonucleoside triphosphate diphosphohydrolase-1 (CD39) [6]. Specifically, upon injury or mechanical stress, intracellular ATP is released to the extracellular space through pannexin/connexin channels [7]. The extracellular ATP is hydrolyzed to ADP and AMP by CD39, and subsequently hydrolyzed to the adenosine metabolite via CD73. In addition, tissue nonspecific alkaline phosphatase (TNAP) has also been shown to contribute to the extracellular adenosine via degradation of ATP to adenosine [8]. Once in the extracellular space, adenosine functions through G-protein-coupled receptors, specifically binding to four distinct adenosine receptors – ADORA1, ADORA2A, ADORA2B, and ADORA3. Different concentrations of adenosine are needed to activate these receptors. At typical physiological adenosine concentration of 0.3–3 nM and 1–20 nM, ADORA1 and ADORA2A can be activated, respectively [9]. Contrarily, ADORA2B and ADORA3 require much higher levels of adenosine, with activation occurring at concentrations of 10 μM and 1 μM, respectively. Although a majority of adenosine is produced from nucleotide metabolism, alternative pathways also contribute to adenosine production. For instance, adenosine can be intracellularly generated from hydrolysis of S-adenosyl-homocysteine (SAH) in the methionine cycle, and subsequently transported to the extracellular space through equilibrative nucleoside transporters (ENT1/2) [4]. Interestingly, molecules other than adenosine, such as Netrin, can also enhance extracellular adenosine signaling. For example, Netrin-1 can activate ADORA2B and has been shown to promote revascularization during limb ischemia [10].

This mini review will discuss how alterations in adenosine signaling contribute to changes in bone health and repair outcomes. Although the significant role played by adenosine in bone homeostasis and its potential therapeutic impact has been widely recognized, severe side effects caused by systemic administration have limited its clinical use. This review will also discuss the technological advancements in the field to circumvent the challenges associated with adenosine delivery.

Adenosine and purinergic signaling in bone health and disease

Adenosine signaling has emerged as a key metabolic pathway that regulates bone tissue formation, function, and homeostasis. Bone tissue-specific cells (osteoblasts and osteoclasts) express all four types of adenosine receptors. Activation of ADORA1 has been shown to be critical for osteoclast differentiation by functioning through tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1) signaling [11]. In fact, ADORA1 knockout mice show significantly higher bone density than wild type mice, which is associated with the loss of ruffled borders of osteoclasts in the knockout mice [12]. ADORA2A activation counters the stimulation of ADORA1, in that it inhibits osteoclastogenesis and osteoclast function [13]. Similarly, ADORA2B activation has been shown to promote osteoblast differentiation and increased function, while also reducing osteoclast numbers [14,15]. ADORA3 has been investigated less compared to other adenosine receptors; however, studies have shown that ADORA3 activation can prevent osteoclast bone resorption [16]. Overall, adenosine receptor activation (or inhibition) is touted to have a major effect on bone homeostasis and bone tissue regeneration (Figure 1). Factors including changes in progenitor cell function, distorted inflammatory response, and reduced neo-vascularization have been implicated in impaired fracture healing [17,18]. Aside from bone-specific cells, adenosine signaling also influences various types of immune cells, both in innate and adaptive immunity. For example, monocytes, macrophages, neutrophils, and B cells express all four adenosine receptors and can be regulated by extracellular adenosine. An abundant source of extracellular adenosine can also be provided by immune cells such as neutrophils and regulatory T cells (Tregs). Adenosine also functions as an anti-inflammatory molecule. ADORA2A and ADORA2B activation have been shown to reduce macrophage production of pro-inflammatory mediators while increasing production of the anti-inflammatory cytokine IL-10 [19,20]. Additionally, adenosine has been shown to polarize macrophages from an M1-like to M2-like phenotype through ADORA2A and ADORA2B [21]. Adenosine, at micromolar concentrations, inhibits recruitment and activation of neutrophils and suppresses effector T cell function while promoting immunosuppressive Tregs [20]. Extracellular adenosine-mediated immunomodulation can also have a significant impact on bone tissue repair, as timely resolution of inflammation is necessary to achieve normal bone healing [22]. Angiogenesis and vascularization can be influenced by immune cells such as macrophages during bone tissue repair. Wang et al. demonstrated that administration of an ADORA2A antagonist altered macrophage secretome, which led to decreased angiogenesis and bone nonunion [23*]. Together these studies suggest that adenosine signaling influences multiple aspects of bone repair, and therefore, adenosine could serve as a potential therapeutic for bone healing.

Figure 1.

Extracellular adenosine generation and signaling in bone health. Extracellular adenosine can originate extracellularly or intracellularly. Most of the extracellular adenosine generation is mediated by ectonucleotidases (CD39 and CD73), which enzymatically degrade adenine nucleotides (ATP, ADP, AMP) to produce adenosine. Within the cell, adenosine can be generated from the methionine cycle where S-adenosyl-homocysteine (SAH) is hydrolyzed. Intracellular adenosine can be transported to the extracellular space through equilibrative nucleoside transporters (ENT1) or converted to ATP through phosphorylation by adenosine kinase (AK), AMP kinase (AMPK), and a combination of nucleoside diphosphate kinase (NDPK) and ATP synthase. The intracellular ATP can be released to the extracellular space through pannexin/connexin (Panx-1/Cx43) channels. Once in the extracellular space, ATP can be metabolized into adenosine via ectonucleotidases. Extracellular adenosine can activate four distinct adenosine receptors – ADORA1, ADORA2A, ADORA2B, and ADORA3. Following activation, these G-protein-coupled receptors can upregulate or inhibit adenylyl cyclase production of cyclic AMP (cAMP) and intracellular signal transduction. This ultimately affects bone cell activity and homeostasis. Adenosine deaminase (ADA) can irreversibly deamidate excess adenosine to inosine.

Multipotent stem cells such as bone marrow mesenchymal stem cells (MSCs) can differentiate to osteoblasts; MSCs can also differentiate into adipocytes and chondrocytes. We have shown that activation of ADORA2B promotes osteogenic differentiation of MSCs while inhibiting their differentiation into adipocytes [24]. Thus, adenosine-mediated osteogenic differentiation of mesenchymal progenitor cells could play a key role in promoting bone tissue regeneration. In fact, Adora2b knockout mice showed a reduction in MSC differentiation to osteoblasts as well as delayed/compromised fracture healing, demonstrating the role of adenosine signaling in bone tissue regeneration [25]. Similarly, Adora2a knockout mice have significantly decreased bone volume and trabecular number. Studies using CD73 knockout mice also show impaired osteoblast differentiation, osteopenia in young animals, and delayed bone regeneration in aged animals [26–28]. Consistent with these findings, a recent study by Kimura et al. showed endogenous CD73⁺ MSCs promote fracture repair and give rise to new cartilage and bone [29*].

Osteoporosis is a metabolic bone disorder characterized by a loss of bone mass. This disease is a result of an imbalance in bone homeostasis with enhanced osteoclastic activity leading to bone resorption. Recently, it has been shown that adenosine signaling is altered in osteoporosis, as CD39 and CD73 expression along with extracellular adenosine levels in the bone marrow are significantly decreased in ovariectomized animals [30]. Aside from changes in bone cell activity and function, increased inflammation and chronic inflammation have also been associated with osteoporosis. These characteristics were complemented by a study that showed animal intake of excessive caffeine, an adenosine antagonist, resulted in increased IL-1β and bone resorption in a periapical periodontitis model [31]. Contrarily, Llamas-Granda et al. have shown that use of molecules like dipyridamole, which augments the effects of extracellular adenosine, reduce bone loss in ovariectomized animals [32*]. Dipyridamole can inhibit equilibrative nucleoside transporter (ENT) uptake into the cell, and thereby increase the extracellular adenosine concentration. Similarly, ticagrelor can also increase extracellular adenosine concentration by functioning as an inhibitor of ENTs [33]. Together, these studies suggest the therapeutic potential of adenosine in combating osteoporosis and improving bone healing.

Harnessing adenosine signaling to promote bone tissue repair

Given that adenosine can contribute to various phases of bone homeostasis and bone regeneration, harnessing adenosine signaling provides a therapeutic opportunity to treat bone disorders. Leveraging adenosine signaling, either through the administration of adenosine or adenosine receptor agonists, could be an effective approach to address bone tissue regeneration; however, such an approach has remained elusive. One limitation is the extremely short half-life of adenosine, which is less than 10 seconds, and thus its reduced bioavailability [34]. Another complication is the severe side effects associated with systemic administration of adenosine, such as suppressed cardiac function, chest discomfort, and dyspnea [35]. A leading concept in drug delivery is utilizing a carrier to improve the delivery efficacy and biodistribution of the drug molecule. Studies have leveraged various drug carriers ranging from liposomes to polymer-based carriers to deliver adenosine [36,37*,38,39*,40*]. Adenosine can be incorporated into the biomaterial and its release can be designed to occur through one or a combination of mechanisms such as hydrolysis, tissue-specific enzymes, or diffusion-mediated release [40*,41,42]. The vicinal diol groups of adenosine offer a unique drug loading opportunity. Specifically, the diol groups can react with boronate groups leading to reversible boronate ester formation. The reversible covalent bond between the adenosine and boronate group allows for both loading and release of adenosine molecules [40*]. Recently, this reversible bond has been utilized to incorporate adenosine into a nanocarrier [37*]. The adenosine delivery units can be further modified to target delivery to the bone tissue. Bone targeting of nanocarriers functionalized with alendronate, which binds to hydroxyapatite minerals, and loaded with adenosine showed therapeutic efficacy in treating osteoporosis in a mouse model [37*,39*]. Since activated osteoclasts release hydrochloric acid and acidify the local niche, hydrogen ions which decrease the pH of the tissue can be used as a trigger for the release of adenosine [39*]. Unlike bisphosphonate-based drugs that only target osteoclast function, adenosine is an osteoanabolic molecule that promotes osteoblastogenesis while inhibiting excessive osteoclastogenesis . Consistent with these understandings, osteoporotic mice treated with adenosine showed new bone formation and bone qualities similar to healthy controls [37*,39*]. Recently, Larrañaga-Vera directly conjugated CGS21680, a selective ADORA2A agonist, to alendronate through a PEG-linker to treat osteoporotic pathology in a mouse model. Osteoporotic mice treated with the ADORA2A agonist exhibited new bone formation and reduced bone loss [43].

While bone disorders like osteoporosis require systemic administration, local delivery of adenosine might be adequate to treat bone injuries such as fractures. Zeng et al., devised a biomaterial approach to capitalize on the surge of extracellular adenosine released following fracture, where a macroporous hydrogel was designed to reversibly sequester the endogenous adenosine to increase both its bioavailability and local signaling [40*]. The results showed that the biomaterial-assisted localization of adenosine at the fracture site promoted vascularization and accelerated bone healing. In a following study, an injectable in situ curing scaffold that enables minimally invasive therapeutic administration, was designed to deliver endogenous adenosine to the fracture site [38]. Studies have also shown that local implantation of a fibrin gel containing an ADORA2A agonist improved bone healing in rat fractures [44]. The authors showed that local delivery of the ADORA2A agonist resulted in an altered inflammatory response of increased Tregs accompanied by decreased Th17 cells and IL-6. Similar to fracture healing studies, critical sized cranial defects treated using a biomaterial loaded with adenosine showed increased bone regeneration [45–47]. Purinergic signaling and increases in extracellular adenosine have also been used to treat several other localized musculoskeletal tissues such as rotator cuff tears, with a focus on increasing tendon-bone junction healing, as well as osteoarthritis, by reducing cartilage degeneration [36,48].

Concluding remarks

In summary, the recent studies discussed complement previous understandings of purinergic signaling in maintaining bone health and repair. These studies clearly demonstrate the key role played by extracellular adenosine, mostly generated by the “classical” adenosinergic pathway involving ectonucleotidases, in bone homeostasis and regeneration. Indeed, alterations in ectonucleotidase function and compromised extracellular adenosine production have been associated with bone metabolic diseases such as osteoporosis and compromised bone regeneration following injury. Bone regeneration is a complex, well-orchestrated physiological process involving multiple cell populations and cellular/molecular processes. Adenosine, which can act on multiple cell populations including immune cells, while simultaneously promoting osteogenesis and angiogenesis, represents a novel and effective therapeutic molecule. Towards this, various drug delivery strategies, both local and systemic, have begun to be explored. Biomaterial-based drug delivery systems, from macroscopic structures to nanocarriers, are not only effective in improving the safety but can also prolong the release kinetics and half-life of adenosine molecules. Although biomaterial-assisted delivery of adenosine is in the early stages of exploration, it has shown promising results in animal models of bone injuries and osteoporosis. Continued development of therapeutic strategies that enable exogenous adenosine delivery and/or activate the purinergic signaling pathway to promote bone health and combat skeletal disease, could potentially translate to clinical treatments.