Purinergic Signaling in Bone

Cancer-induced bone pain severely compromises the quality of life of many patients su ﬀ ering from bone metastasis, as current therapies leave some patients with inadequate pain relief. The recent development of speciﬁc animal models has increased the understanding of the molecular and cellular mechanisms underlying cancer-induced bone pain including the involvement of ATP and the purinergic receptors in the progression of the pain state. In nociception, ATP acts as an extracellular messenger to transmit sensory information both at the peripheral site of tissue damage and in the spinal cord. Several of the purinergic receptors have been shown to be important for the development and maintenance of neuropathic and inﬂammatory pain, and studies have demonstrated the importance of both peripheral and central mechanisms. We here provide an overview of the current literature on the role of purinergic receptors in cancer-induced bone pain with emphasis on some of the di ﬃ culties related to studying this complex pain state. Modulation of tumor microenvironment by di ﬀ erent mediators is central in determining neoplastic formation and progression. Among these molecules extracellular ATP is emerging as a good candidate in promoting cell growth, neovascularization, tumor-host interactions, and metastatization. This paper summarizes recent ﬁndings on expression and function of P2X7 receptor for extracellular ATP in primary and metastatic bone cancers. Search of mRNA expression microchip databases and literature analysis demonstrate a high expression of P2X7 in primary bone tumors as well as in other malignancies such as multiple myeloma, neuroblastoma, breast, and prostate cancer. Evidence that P2X7 triggers NFATc1, PI3K/Akt, ROCK, and VEGF pathways in osteoblasts promoting either primary tumor development or osteoblastic lesions is also reported. Moreover, P2X7 receptor is involved in osteoclast di ﬀ erentiation, RANKL expression, matrix metalloproteases and cathepsin secretion thus promoting bone resorption and osteolytic lesions. Taken together these data point to a pivotal role for the P2X7 receptor in bone cancer biology. The purinergic P2X7 receptor is expressed by bone cells and has been shown to be important in both bone formation and bone resorption. In this study we investigated the importance of the genetic background of the mouse strains on which the P2X7 knockout models were based by comparing bone status of a new BALB/cJ P2X7 − / − strain with a previous one based on the C57BL/6 strain. Female four-month-old mice from both strains were DXA scanned on a PIXImus densitometer; femurs were collected for bone strength measurements and serum for bone marker analysis. Bone-related parameters that were altered only slightly in the B6 P2X7 − / − became signiﬁcantly altered in the BALB/cJ P2X7 − / − when compared to their wild type littermates. The BALB/cJ P2X7 − / − showed reduced levels of serum C-telopeptide fragment (s-CTX), higher bone mineral density, and increased bone strength compared to the wild type littermates. In conclusion, we have shown that the genetic background of P2X7 − / − mice strongly inﬂuences the bone phenotype of the P2X7 − / − mice and that P2X7 has a more signiﬁcant regulatory role in bone remodeling than found in previous studies. Macrophages from mouse strains with the naturally occurring mutation P451L in the purinergic receptor P2X7 have impaired responses to agonists (1). Because P2X7 receptors are expressed in bone cells and are implicated in bone physiology, we asked whether strains with the P451L mutation have a di ﬀ erent bone phenotype. By sequencing the most common strains of inbred mice, we found that only a few strains (BALB, NOD, NZW, and 129) were harboring the wild allelic version of the mutation (P451) in the gene for the purinergic receptor P2X7. The strains were compared by means of dual energy X-ray absorptiometry (DXA), bone markers, and three-point bending. Cultured osteoclasts were used in the ATP-induced pore formation assay. We found that strains with the P451 allele (BALB/cJ and 129X1/SvJ) had stronger femurs and higher levels of the bone resorption marker C-telopeptide collagen (CTX) compared to C57Bl/6 (B6) and DBA/2J mice. In strains with the 451L allele, pore-formation activity in osteoclasts in vitro was lower after application of ATP. In conclusion, two strains with the 451L allele of the naturally occurring mutation P451L, have weaker bones and lower levels of CTX, suggesting lower resorption levels in these animals, which could be related to the decreased ATP-induced pore formation observed in vitro . The importance of these ﬁndings for the interpretation of the earlier reported e ﬀ ects of P2X7 in mice is discussed, along with strategies in developing a murine model for testing the therapeutic e ﬀ ects of P2X7 agonists and antagonists upon postmenopausal osteoporosis. P2X7 receptor-mediated purinergic signaling is a well-known mechanism involved in bone remodeling. The P2X7 receptor has been implicated in the pathophysiology of various bone and cartilage diseases, including rheumatoid arthritis (RA), a widespread and complex chronic inﬂammatory disorder. The P2X7 receptor induces the release into the synovial ﬂuid of the proinﬂammatory factors (e.g., interleukin-1 β , prostaglandins, and proteases) responsible for the clinical symptoms of RA. Thus, the P2X7 receptor is emerging as a novel anti-inﬂammatory therapeutic target, and various selective P2X7 receptor antagonists are under clinical trials. Extracellular ATP signaling acting through the P2X7 receptor is a complex and dynamic scenario, which varies over the course of inﬂammation. This signaling is partially modulated by the activity of ectonucleotidases, which degrade extracellular ATP to generate other active molecules such as adenosine or pyrophosphates. Recent evidence suggests di ﬀ erential extracellular metabolism of ATP during the resolution of inﬂammation to generate pyrophosphates. Extracellular pyrophosphate dampens proinﬂammatory signaling by promoting alternative macrophage activation. Our paper shows that bisphosphonates are metabolically stable pyrophosphate analogues that are able to mimic the anti-inﬂammatory function of pyrophosphates. Bisphosphonates are arising per se as promising anti-inﬂammatory drugs to treat RA, and this therapy could be improved when administrated in combination with P2X7 receptor antagonists.

In recent years, it has become apparent that extracellular nucleotides, signalling via P2 receptors, play an important role in the regulation of bone turnover. Furthermore, purinergic signalling has been associated in the pathophysiology of several bone and cartilage diseases, including osteoarthritis, rheumatoid arthritis, and osteoporosis and might also be implicated in the deleterious skeletal effects of cancer and on bone pain. Widespread expression of multiple P2 receptor subtypes by bone and cartilage cells has now been reported, and the functional effects of receptor activation are being determined. Of all of the P2 receptors expressed, it is the P2X7 receptor which has emerged as being central in the pathogenesis of several skeletal conditions, though also a number of other P2Y and P2X receptors have important roles in regulation of bone turnover.
There are many studies, using human and animal models, which have described the pivotal role of the P2X7 receptor in rheumatoid arthritis, a complex, multifactorial inflammatory disease with no current successful treatment. The review entitled "Modulating P2X7 receptor signaling during rheumatoid arthritis: new therapeutic approaches for bisphosphonates" by A. Baroja-Mazo and P. Pelegrin included in this special issue summarises some of this research. In particular, it focuses on the therapeutic potential of P2X7 receptor antagonists, both alone and in combination with bisphosphonates, as a treatment for rheumatoid arthritis.
Recent work has shown that the P2X7 receptor is also important in bone-related conditions, particularly osteoporosis. A number of clinical studies have associated singlenucleotide polymorphisms (SNPs) in the P2X7 receptor gene with increased fracture risk, low bone mineral density, and increased bone loss in humans. The importance of genetic variation within the P2X7 receptor in relation to bone mass and strength is addressed by two original research articles by S. Syberg et al. in this special issue. Firstly, the paper entitled "Association between P2X7 receptor polymorphisms and bone status in mice" investigates the effects of one SNP (P451L) using an in vivo animal model. The second article entitled "Genetic background strongly influences the phenotype of P2X7 receptor knockout mice" provides a detailed analysis of the differences of bone phenotype between two different strains of P2X7 receptor knockout. Combinedly these research papers highlight the importance of genetic background when looking at the functional effects of the P2X7 receptor and suggest that when mouse models are used to test the efficacy of P2X7 receptor agonists and antagonists it is taken into account. Also they demonstrate the role of the P2X7 receptor in regulation of bone mass.
Bone is both a site of primary tumor formation and metastatic spread of high incidence neoplasias such as breast and prostate cancers. To date, the efficacy of therapies intended to reduce bone alterations and related pain in cancer is limited. In this special issue, two papers point out to P2X receptors as possible targets for the treatment of bone cancer and associated pain. E. Adinolfi et al. cover recent findings linking P2X7 receptor and bone biology with a focus on P2X7-mediated osteoblast proliferation and osteoclast differentiation. The authors report evidence on the role of the P2X7 receptor as an oncogene implicated in cancer growth, neovascularization, and metastatic dissemination.
Their paper relies on data reported by the recent literature and examination of Affymetrix-based expression databases. Examined tumors include osteosarcoma, neuroblastoma, multiple myeloma, and breast and prostate cancers.
The review from S. Falk et al. is centered on the role of purinergic nociception in cancer-induced bone pain. In particular, the authors report evidence in favor of P2X3 involvement in bone-related pain both at the peripheral and central levels concentrating on studies conducted in different rodent models of cancer-induced pain states. The authors also report on the role of other P2X and P2Y receptors in the complex network of cells involved in bone pain development, with a critical perspective taking into account all the possible problems linked to model representative potential. "P2X7 receptor function in bone-related cancer" and "The role of purinergic receptors in cancer-induced bone pain" both take into consideration the role of the natural agonist of P2X receptors, extracellular ATP, in cancer. Indeed, several publications reported the presence of high levels of ATP in cancer microenvironment, and a role for this nucleotide in increasing the host immune response was suggested. Data summarized in the reviews by E. Adinolfi et al. and S. Falk et al. suggest a mechanism by which extracellular ATP released from tumor cells, through the P2X7 receptor, might affect osteoblast growth and osteoclast activity, while it might induce pain through the P2X3 receptor.
Increasing evidence supports the role of purinergic signaling through P2 purinergic receptors in regulating normal bone turnover, but also seems to play a role in the pathophysiology of a range of bone diseases including postmenopausal osteoporosis, immune-mediated bone loss, and cancer-induced bone disease as well as in bone-pain.
Thus, accumulating evidence provide us with a range of new therapeutic targets to treat the above-mentioned diseases, for some of which efficacious treatment options are not currently available. In terms of therapeutic strategy for cancer-induced bone disease, one could speculate that treatment with a cocktail of drugs contemporarily targeting multiple P2X receptor could prove efficacious both in reducing cancer growth, dissemination and pain sensation, while the widespread expression of P2 receptors on the different types of bone cells could prove to be a novel target for the regulation of bone formation and resorption in both postmenopausal osteoporosis and osteoporosis related to other diseases.
It will certainly be interesting to follow the continued progress in the field of purinergic signaling in bone in the future, and the growing acceptance of this concept is supported by the recent funding by the European Commission's 7th Framework Programme of the ATPBone project and a dedicated session at the European Calcified Tissue Society 2012 conference to "ATP and bone, " putting this area of research concept on the scientific map.