How purinergic signaling affects bone homeostasis.
M. Zuccarini, M. Carluccio, P. Giuliani and P. Di Iorio
Department of Medical, Oral and Biotechnological Sciences, “G. d’Annunzio” University of Chieti-Pescara, Chieti, Italy
Dr. M. Zuccarini,
Department of Medical,
Oral and Biotechnological Sciences,
“G. d’Annunzio” University of Chieti-Pescara,
Growing evidence suggests that purinergic signaling could play a key role in osteoblast, osteoclast, and chondrocyte function. Osteoblasts and osteoclasts express P1 and P2 receptors that are known to participate in both osteoblast and osteoclast differentiation, thus contributing to bone formation or resorption, respectively. It is widely accepted that ATP and other nucleotides, acting via P2 receptors, contribute to the fusion of osteoclasts by inducing several pro-resorptive factors (i.e. RANK, NFkB, TRAF6). By contrast, adenosine can stimulate osteoblastogenesis via A2A,B-mediated activation of cAMP/PKA signaling pathway. Among others, the role of P2X7 receptor in bone cell function is still controversial. This work discusses the current understanding of the involvement of purinergic signaling in bone development and homeostasis, and sheds light on their use as potential targets for skeletal regenerative therapies.
Key words: purine receptors, purinergic signaling, bone metabolism, RANKL/OPG ratio
Adenine and guanine-based purines participate in a wide range of biological processes, such as neurotransmission and cell metabolism, proliferation and differentiation, in both neuronal and non-neuronal tissues, acting as extracellular signaling molecules via specific receptors (1-3). Extracellular nucleotides are released under both physiological and pathophysiological conditions and then metabolized to the corresponding nucleosides and nucleobases through the concerted activity of ecto- and soluble purine-converting enzymes (4, 5). Purinergic receptors have been widely investigated in the context of bone remodeling and development as they regulate, together with other endocrine and paracrine factors, the fine-tuned activity of osteoclasts and osteoblasts (6). Alterations in the balance between bone forming and bone resorbing processes may disrupt bone development and remodeling, leading to the onset of skeletal disorders and degenerative diseases such as osteoporosis or osteoarthritis.
Purine receptors are divided into two families: P1 receptors, responding to adenosine, and P2 receptors responding to nucleotides. P2 family is further classified in P2X1-7 ionotropic receptors (ligand-gated ion channels) and P2Y1, 2, 4, 6, 11-14 metabotropic/G protein- coupled receptors (7). P2X and P2Y receptors are present on osteoclasts, osteoblasts, and chondrocytes. Upon nucleotide binding, they are able to transiently increase [Ca2+]i and induce inositol (1,4,5)-trisphosphate formation in osteoblast-like cells. Importantly, they have been correlated to adipogenic and osteogenic differentiation of human mesenchymal stem cells, thus giving rise to a possible role in the regulation of bone cell formation and function (8). ATP released from bone cells (e.g., through shear stress or constitutively) can be degraded to adenosine 5′-diphosphate (ADP) or converted into uridine 5′-triphosphate (UTP) through ecto-nucleotidases. In particular, ATP and ADP are able to induce the formation and resorptive activity of osteoclasts as well as reduce bone formation by osteoblasts, acting via P2Y1 and P2X2 receptors (9). ADP could also stimulate resorption indirectly through actions on osteoclasts, which in turn release pro-resorptive factors (e.g., receptor activator of nuclear factor kB ligand (RANKL), a critical osteoclast differentiation factor).
Similarly to ATP or ADP, extracellular UDP, via P2Y6 receptors, has been shown to stimulate the formation of osteoclasts from precursor cells and increase the proresorptive effects on mature osteoclasts. Many reports also suggested a role for purine-converting enzymes in bone mineralization. In this regard, osteoblastic E-NPP activity was shown to produce high concentrations of PPi, a potent inhibitor of bone mineralization (10), thus raising the possibility that ATP- and UTP-mediated inhibition of osteogenesis might rely on their signaling via the P2Y2 or P2Y4 receptors or on a direct hydrolysis to PPi.
Among others, P2X7R represents one of the most controversial receptors in the process of osteogenesis, being able either to promote (11) or to inhibit (12) bone mineralization in osteoblasts. Indeed, the expression and function of P2X7R vary depending on the stage of bone cells (13). Functional P2X7 receptors are expressed by human osteoblasts as well as several other populations of primary human bone-derived cell (hBDC) types. Upon ATP binding, P2X7 causes the activation of MAPKs and the activating protein-1 (AP-1) transcription factors, JunB, c-Fos, FosB, as well as the release of inflammatory cytokines such as IL-1β, or the activation of caspases (14). The AP-1 transcription factors, in addition to runt-related transcription factor 2 (Runx2/Cbfa1), distalless homeobox 5 (Dlx5), mouse segment homeobox 2 (Msx2) and Osterix (Osx), are key players in osteoclast and osteoblast formation (15). These gene regulatory proteins are known to be regulated by several components of the Wnt signaling pathway involved in bone formation. Wnt signaling occurs via both canonical (Wnt3a/β-catenin) and non-canonical signaling pathways. It has been reported that BzATP, a P2X7R agonist, inhibited β-catenin nuclear translocation, significantly decreased ALP activity in primary rat calvarial osteoblasts (12) and reduced nodule formation by primary human trabecular osteoblasts (16). Moreover, P2X7R activation inhibits human mandibular-derived osteoblast differentiation by down-regulating the canonical Wnt signaling pathway. Surprisingly, P2X7R was recently found to have a stimulating, rather than an inhibiting, effect on in vitro mineralization (17) and Wnt/β-catenin signaling (18). In that case, P2X7R activation would induce plasma membrane blebbing and bone formation via potentiation of Wnt/ β-catenin transcriptional activity, production of lipid mediators or FosB-dependent COX-2 protein expression, lypophosphatidic acid (LPA) and prostaglandin E2 (PGE2) (11). In patients with nonunion bone fractures, shockwave therapy enhanced bone healing via ATP release from hMSCs and the subsequent activation of P2X7 receptors responsible for hMSCs osteogenic differentiation and osteoblasts proliferation and mineralization. The molecular mechanism underlying this beneficial effect would include calcium mobilization and the activation of p38 MAPK/c-Jun/c-Fos/AP-1 signaling cascade (19).
Interestingly, several P2 receptor polymorphisms and P2 receptor knockout mice models have been correlated to altered bone development such as skeletal deformities and increased risk of osteoporosis. For example, an association was shown between the P2Y2 L46P polymorphism and increased BMD in the hip, lumbar spine and femoral neck (20), as well as increased adipogenic, rather than osteogenic, differentiation in P2Y13 knockout-MSCs due to changes in phosphate metabolism and hormone levels (21). Furthermore, osteoclasts derived from P2Y6 receptor-deficient mice (P2Y6R-/-) showed decreased resorptive activity parallel to an increased bone mineral content and cortical bone volume, whereas trabecular bone parameters (volume, thickness and number) were unaffected (22). In addition to P2Y6, also P2X7, P2Y1, P2Y2 and P2Y13 knockout mice have been associated to skeletal abnormalities (23). Notably, skeletal analysis of P2Y2 knockout mice by dual energy X-ray absorbtiometry and micro-CT revealed a significant increase in trabecular and cortical bone parameters in both the femora and tibae (10). Recently, strong evidence from studies of extracellular matrix alterations in epithelial-to-mesenchymal transition and cell metastasis (24, 25) supports the hypothesis that purinergic system may also regulate extracellular matrix mineralization, being involved in osteogenic differentiation, tissue mineralization and calcium deposition in the extracellular microenvironment. Noteworthy, it was demonstrated that the Mg2+/Ca2+ balance plays a central role in P2R-mediated extracellular matrix calcification. In this regard, Mg2+ exerts an inhibitory effect on matrix calcification by preventing ATP release and consequent P2R activation (26).
In addition to P2 receptors, a role for adenosine and its receptors in bone and cartilage homeostasis has been suggested (27-29). Human osteoprogenitor and mesenchymal stem cells express all P1 adenosine receptors, which are able to secrete inflammatory cytokine IL-6 and osteoprotegerin (OPG), one of the most known compounds responsible of osteoclastogenesis (30). Osteoclast differentiation is controlled by osteoblasts through expression of RANKL and OPG, a soluble receptor of RANKL able to inhibit RANKL interaction with its receptor, fundamental for the bone mass. In particular, A1AR activation was shown to stimulate osteoclast differentiation. Indeed, A1R selective antagonist, rolofylline, inhibited M-CSF/RANKL- or TNF receptor associated factor 6 (TRAF6) and TAK1 kinase-induced osteoclastogenesis of bone marrow cells. This effect is considered of particular interest when related to ovariectomy-induced bone loss (31). By contrast, A2AR activation displays opposite effects as compared to A1AR, being able to inhibit osteoclast differentiation and enhance bone density, likely through disruption of M-CSF/RANKL, decrease of IL-1β and TNF-alpha levels, and inhibition of cAMP/PKA/ERK1/2 signaling pathway (32). In disagreement with these data, another group found that A2AR activation potentiated M-CSF/RANKL fusion in stimulated human peripheral blood monocytes (33). In patients with rheumatoid arthritis, weekly low-dose methotrexate (MTX) injections decreased bone pitting and increased bone volume, an effect mediated by adenosine-dependent regulation of RANKL/OPG ratio (34). This drug was also shown to inhibit ALP activity in MC3T3-E1 and rat bone marrow stromal cells. In inflammatory bone diseases, cAMP/PKA signaling activated by stimulation of A2AR/A2BR was responsible for osteolysis inhibition and enhanced osteoblast differentiation, whereas an opposite effect was elicited by A1AR. The stimulation of osteoblast differentiation has been associated to the up-regulation of Runx2 expression, a key transcription factor in osteogenesis. A2BR knockout mice showed decreased osteogenesis, bone density and delayed fracture repair (35). Similarly to A1AR, A3AR activation has been correlated to decreased osteoclast differentiation due to PI3K, NFkB, and RANKL downregulation (36). Intriguingly, adenosine seems to affect cartilage metabolism, as its depletion accounted for joint destruction and inflammation, due to increased production of glycosaminoglycan (GAG), matrix metalloproteinases
(MMP-3, MMP-13), prostaglandin E2 and nitric oxide (NO) (37). In fact, treatment of murine chondrocytes with a selective A2AR agonist, CGS-21680, was able to decrease several inflammatory markers such as TNF-alpha, IL-6, MMP-13 and NO, and the application of this compound to arthritic mice reduced paw edema and joint destruction (38). Accordingly, a μ-CT analysis of A2AR KO mouse femurs showed a significantly decreased bone volume/trabecular bone volume ratio, decreased trabecular number, and increased trabecular space. Cumulative evidence suggests that adenosine-converting enzymes are crucial players in cartilage-related processes. In particular, increased adenosine deaminase (ADA), an enzyme that catalyzes adenosine conversion to inosine, has been found in the synovial fluid of patients affected by rheumatoid arthritis and systemic lupus erythematous (39). Moreover, patients with ADA deficiency presented skeletal abnormalities including unusual scapular spurring and anterior rib cupping. Surprisingly, the adenosine-generating enzyme (ecto-5’-nucleotidase) has been positively correlated to the progression of osteoarthritis, displaying a higher activity in disrupted cartilage, although other studies reported that CD73 knockout mice were osteopenic (40).
Overwhelming experimental evidence points to a significant role of purinergic signaling in bone cell function and skeletal mechanotransduction, taking part in the dynamic and complex processes accounting for bone remodeling (Fig. 1). In this scenario, extracellular purines and their specific receptors may represent promising targets for the development of novel drugs capable of preventing bone resorption, promoting osteogenesis and accelerating fracture healing in those diseases characterized by cartilage erosion and joint alterations, such as rheumatoid arthritis, tendinosis, osteoporosis and tumor-induced osteolysis. Noteworthy, several purine receptor agonists and antagonists have already been used in clinical trials such as IB-MECA (CF101, developed by Can-FiteBioPharma), an A3AR agonist that has been tested on patients with rheumatoid arthritis, psoriasis and dry eye disease. Given these observations and the availability of more KO models, further analysis of the role of purinergic receptors in bone homeostasis in health and disease is clearly warranted.
Fig. 1. Schematic representation of purinergic signaling in bone homeostasis. ATP is released from osteoblast-derived cells and metabolised into adenosine 5′-diphosphate (ADP) or converted into uridine 5′-triphosphate (UTP). ATP, ADP and UTP interact with specific P2 receptor subtypes (i.e. P2Y2 and P2Y4 receptors) and stimulate the fusion of osteoclasts and the activation of pro-resorptive factors (i.e. RANKL, NFkB, IL6, TRAF6). The role of P2X7R signaling in bone is still controversial. Upon ATP binding, P2X7R activation stimulates either bone formation by osteoblasts or cell death of mature osteoclasts, through inhibition of Wnt/β-catenin-induced Runx2 and OSX trascription. At extracellular level, ATP is also converted by ecto-nucleotidases into adenosine that, in turn, interacts with P1 receptors and enhances (A2A,B) or inhibits (A1,3) osteblast differentiation mainly via cAMP/PKA signaling.
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