Final Report Summary - ATPBONE (Fighting osteoporosis by blocking nucleotides: purinergic signalling in bone formation and homeostasis)
Osteoporosis is a widespread systemic disorder characterised by decreased bone mass and deterioration of bone micro-architecture. It results from a combination of genetic, environmental and other risk factors and is a consequence of an unbalance of bone formation and bone resorption. In view of the rapidly increasing prevalence of osteoporosis in European societies, it is of major importance to create new and more effective pharmaceuticals for fighting osteoporosis in the future.
Purinergic signalling, where ATP and its metabolite adenosine act as extracellular signalling molecules is a rapidly expanding field. Evidence has accumulated that the purinergic system plays a central role in bone physiology. Thus, the overall aim of the present project is to provide mechanistic basis for nucleotide-based therapeutics in osteoporosis. For this purpose, we have gathered in the ATPBONE consortium the main research groups in the European Union (EU) working in the field of osteoporosis and purinergic signalling. Participating institutions are Copenhagen University Hospital Glostrup, Denmark; University College London, United Kingdom (UK); University of Maastricht, the Netherlands; University of Ferrara, Italy; University of Liverpool, UK; University of Sheffield, UK; and Université Libre de Bruxelles, Belgium. The project was funded with EUR 3 million through the EU Seventh Framework Programme (FP7), and commenced 1 January 2008 with a duration of 3 years.
The work plan included:
(A) basic in vitro investigations on ATP release and signalling;
(B) a translational section involving in vivo experiments including transgenic mice,;and
(C) studies of human cohorts and clinical intervention studies.
Thus, the project involved on the one hand a fundamental part, aiming to give a basic description of the purinergic signalling system, and on the other hand a translational part to demonstrate the importance of the purinergic system in osteoporosis treatment. Multiple experimental approaches have been made, including physiological challenge of bone metabolism by mechanical loading, and pharmacological intervention on the purinergic signalling system.
The results have been highly interesting and encouraging. In the consortium, we have obtained a very detailed insight into the basic mechanisms of purinergic signalling in bone, as well as a better understanding of the regulation of bone metabolism including the formation of new bone, degradation of old bone tissue as well as the mineralisation of bone matrix. The in vivo experiments have given us important new knowledge of the roles of the P2 purinergic receptors on the regulation on bone turnover in the living skeleton, and in the translational studies we have provided new and significant evidence that genetic variations in the genes coding for the P2 receptors are associated with increased bone loss, osteoporosis and fragility fractures, indicating an important role of P2 receptor defects in osteoporosis pathophysiology. Finally, the highly encouraging early pre-clinical studies in mice have provided evidence for the P2 receptors as pharmaceutical targets for anti-osteoporotic treatments, which will be pursued further within the frames of the consortium even after the project is officially closed.
Thus, the project has provided both mechanistic insight into basic mechanisms of bone turnover as well as into osteoporosis pathophysiology, and the role and importance of the P2 receptors in bone metabolic diseases will be further exploited in future projects within the consortium.
For more information on the project and the consortium, please see the ATPBONE webpage at http://www.ATPBone.eu or contact scientific coordinator Niklas Rye Jørgensen (niklas@dadlnet.dk via mail).
Project context and objectives:
Osteoporosis is a widespread systemic disorder characterised by decreased bone mass and deterioration of bone micro-architecture. The social economic burden is high as it is estimated that in Europe about 1 in 3 women and 1 in 8 men above the age of 50 will suffer an osteoporotic fracture. These numbers are expected to increase exponentially worldwide, as the ageing population increases significantly and the sedentary lifestyle and changes in nutritional habits are making the impact even larger. Osteoporosis results from a combination of genetic, environmental and other risk factors that affect both peak bone mass and the rate of bone loss. These factors include gender, age, age at menopause, use of glucocorticoids, chronic disease, diet, race, lifestyle and physical activity. Osteoporosis may be either primary or secondary. Bone loss is a result of a dysbalance of bone formation and bone resorption. In postmenopausal osteoporosis, which is the most common form, there is evidence that bone resorption is increased without sufficient compensatory increase in bone formation. Effective anti-resorptive therapy is available to reduce the risk of osteoporotic fractures: Calcium and vitamin D, selective estrogen-receptor modulators, bisphosphonates and strontium ranelate. However, even though these agents reduce bone resorption, they have little, if any, effect on bone formation. Parathyroid hormone (PTH) treatment is anabolic and may be used in very severe cases of osteoporosis for up to one an a half year in European countries, leading to increased bone quality and strength. However, PTH is currently the only anabolic agent for osteoporosis treatment and with a limited treatment period of use due to the possibility of stimulating osteosarcoma in patients. In view of the high disease load of osteoporosis and its expected dramatic rise over the next decades, as well as the very limited accessibility to anabolic treatments it is pressing to create new effective pharmaceuticals for fighting osteoporosis in the future. For this purpose, it is of major importance to elucidate the currently ill-understood basic mechanisms of bone metabolism and function on a cellular level particularly receptor signalling and pathways involved in the normal osteoclast and osteoblast function.
Purinergic signalling, where ATP and its metabolite adenosine act as extracellular signalling molecules in a whole range of tissues, is a rapidly expanding field. Over the past decade, evidence has accumulated that the purinergic system plays a central role in bone physiology. Osteoblast and osteoclast physiology is finely tuned by extracellular nucleotides, and so are bone deposition and resorption. Signalling by extracellular ATP in bone is even more fascinating due to the exquisite mechanical properties of this tissue, and to the known ATP-releasing ability of mechanical stresses and tensions, to the point that it is now widely believed that release of ATP from bone cells is one of the main transduction pathways of mechanical stimulation. ATP has also been recognised as one of the most important early signalling molecules released by injured cells during the early phases of inflammation also known as danger associated molecular patterns (DAMPs), while adenosine has an well-established role as a potent immunosuppresive factor. Like most immuno-modulating agents, ATP can act either as an immunosuppressive or an immunostimulatory factor, depending on the dose, the cytokine microenviroment and the pattern of P2 receptor expression of the target cells. This may be particularly relevant in the bone microenvironment where nucleotides may modulate expression of membrane or soluble receptors involved in osteoblast/osteoclast maturation, such as receptor activator of nuclear factor kappa (RANK) and its ligand (RANKL) or osteoprotegerin, and may enhance sensitivity of bone cells to hormones such as PTH.
The overall aim of the proposal, based on current knowledge of the purinergic signalling system and our previous work in the field, is to provide mechanistic basis for nucleotide-based therapeutics in osteoporosis, effective through modulation of bone formation and homeostasis. The large number of different P2 receptor subtypes with widely differing affinity for extracellular nucleotides and the many different biologic responses that can be elicited by stimulating each of them, makes the 'purinergic system' a highly flexible target for the design of innovative molecules for the treatment of bone disease.
Project results:
Overall strategy of work plan
The proposed project was designed as a multidisciplinary approach to elucidate the role of ATP signalling through P2 receptors on bone with special emphasis on the effects on bone formation and homeostasis. The work plan included:
A) fundamental in vitro investigations on ATP release and signalling;
B) a translational section using animal experiments including transgenic mice; and
C) studies of human cohorts and clinical intervention studies.
Thus the ATPBONE project aimed at giving both a basic description of the purinergic signalling system and a translational part demonstrating the value of the system in osteoporosis treatment in a highly interrelated and integrated project. Interventions included both mechanical loading, as the normal physiologic stimulus for bone formation, and pharmacological intervention in the purinergic signalling system. Potential interactions of the purinergic signalling system with other established and novel factors affecting bone metabolism, such as oestrogen, parathyroid hormone, prostaglandins, and mediators of inflammation and oxidative stress, were examined.
ATP as a signalling molecule
The objective of the first part of the project was to describe the signalling molecule, ATP, in terms of demonstrating:
a) its mechanisms of release from bone cells; and
b) the stimuli leading to enhancement of release.
Particular emphasis was paid to the effects of mechanical stimulation, since this is an important stimulus of bone growth. The mechanically induced ATP release was tested both in vitro and in vivo partially using a new transgenic mouse created within the consortium. This mouse expresses plasma membrane-bound recombinant luciferase (pmeLUC) in order to visualise in vivo ATP release. The effect of other physiologically relevant stimuli such as hydroxyapatite and hypoxia were be tested. Since nucleotides are rapidly degraded by enzymes and the effect of nucleotides on bone cells is therefore influenced by the degradation and conversion patterns, the initial work also addressed the mechanisms for degradation and conversion of nucleotides.
During the work on this objective, we demonstrated that bone forming cells (osteoblasts) in cultures release ATP constitutively. Mechanical stimulation in the form of fluid displacement stimulates the release of ATP from osteoblasts, which was confirmed in three different model systems. Three types of mechanical loading have been applied, fluid displacement, shaking and vibration. Results have demonstrated that fluid displacement, which generates fluid shear forces, consistently enhances the release of ATP into conditioned medium and is a better stimulus than shaking or vibration. This indicates that fluid flow might be the most physiological stimulus in vivo. Also the substrate on which the cells are attached influences the release of ATP, and the response to fluid shear. Cells cultured on calcified substrates tend to release more ATP than cells on plastic culture plates. These studies have also revealed that ATP can bind to mineralised matrix. Also the bone resorbing cells, osteoclasts, were shown to be able to release ATP when stimulated mechanically.
To investigate whether ATP is released from bone in vivo, we engineered a mouse expressing a reporter for ATP release, also called pmeLUC. Examining this mouse in a whole body luminometer enables the detection of ATP release in living animals. Generation of this transgenic animal has requested a considerable effort, that however was well spent as the pmeLUC mouse appears to be a very sensitive detector of ATP release into the extracellular space, and therefore of mechanical stimulation, tissue damage or inflammation. Along the same lines, we have further developed the HEK293-pmeLUC biosensor that was at the basis of the construction of the pmeLUC-mouse. We can now safely conclude that the HEK293-pmeLUC biosensor is a very sensitive and reliable indicator of changes in the extracellular ATP concentration in a number of different conditions ranging from mechanical stimulation to cancer, from graft-versus-host to contact sensitivity reactions. Lack of a reliable sensor for the measurement of extracellular ATP has long been a major drawback in the purinergic receptor field, and the engineering of the pmeLUC probe is a real break-through. One of the main objectives was to determine whether ATP is release in bone upon mechanical stimulation in vivo. We therefore applied a new model of bone loading. Traditional in vivo models of bone loading have relied on passive loading. We have developed a novel method which relies on muscle activation. Stimulator devices are implanted in the animals stimulating the nerve to one hindlimb, causing muscular contraction and subsequent loading of bones. By using this model we have demonstrated release of ATP in vivo in response to loading. Furthermore, we have shown that mechanical loading induces changes in expression of multiple genes in muscle and in bone.
To further study which factors other than mechanical forces that can induce ATP release in bone cells, we examined a range of stimuli that are known to affect nucleotide release from other cell types. We found that low oxygen tension (hypoxia) as well as other types of mechanical stimulation could induce ATP release from the bone cells.
The last part of the studies on ATP focused on the regulation of ATP concentrations outside the cells. Once released the activity of nucleotides are limited by the actions of enzymes which convet and degrade nucleotides to their corresponding nucleoside and phosphate. We have studied the expression of these enzymes on osteoblasts under normal and acidic conditions. We have shown that osteoblasts express multiple enzymes and that the expression of these enzymes changes during osteoblast differentiation. Work is ongoing to investigate other factors which regulate the expression and activity of these enzymes.
Expression and signalling pathways of P2 purinergic receptors
There are a number of different P2 purinergic receptors belonging to the two subgroups, P2Y and P2X, These are clearly distinct in nature and in order to understand the functions of the purinergic signalling system in bone, it is important to determine the expression of P2 receptors in bone cells as well as the effects of receptor activation on bone formation and degradation. In addition, hormones (oestrogen and parathyroid hormone) and other factors that regulate bone turnover, might interact with the P2 receptors' functions. Therefore, the next part of the project focused on investigating the exact expression, functions and interactions with hormones of the individual and distinct P2 receptors.
During the work on this objective, we described the detailed expression of P2 receptors on bone cells, including the on the bone forming osteoblast and on the bone degrading osteoclasts. We also showed that the receptors play significant roles in bone, modulating both osteoblast and osteoclast function and that the expression pattern changes during the differentiation of the cells. We also showed that stimulation or inhibition of the different receptors with universal or specific agonists and antagonists could modulate the activity of the bone cells so that bone forming activity could either be stimulated or inhibited depending on the specific receptor targeted. We also used cells from mice that did not express single specific receptors, confirming the findings from the agonist / antagonist studies.
As parathyroid hormone is the most important regulator of bone metabolism in vivo, it is highly relevant to investigate whether there is an interaction between parathyroid hormone signaling a dn purinergic signaling. We have previously shown that dual activation of P2 receptors and parathyroid hormone receptors on bone forming cells leads to a synergistic action. The studies demonstrated that transient exposure of bone cells to ATP sensitises the cells to the action of parathyroid hormone for up to nine hours. Furthermore, the parathyroid hormone and ATP also interacts on genes including that regulates the formation and activity of the bone resorbing osteoclasts. This synergy suggests a mechanism whereby locally released extracellular nucleotides can enhance the sensitivity of the cells to systemic hormones and thereby activate bone remodelling at target sites. We have extended these observations to other systemic hormones including the gut hormones, glucose-dependent insulinotropic polypeptide (GIP) and the adipocyte-derived hormone, adiponectin.
One of the subtypes of the P2 receptors has received special attention. It is the P2X7 receptor that has distinct features compared to the other P2X receptors as it can form a non-selective and large conductance pore. There has been a long-standing issue of the participation of pannexin-1 to the generation of the P2X7 pore. Our data provide unequivocal demonstration that pannexin-1 is not a constituent of the P2X7 pore, but we have found that pannexin-1 has a key role in ATP release, In addition we have performed an extensive investigation of the down-hill signalling molecules from the P2X7 receptor.
The P2X7 receptor is highly variable and we have therefore further investigated this. We have cloned and fully characterised a novel truncated splice variant of the human P2X7 receptor named P2X7B. This isoform is expressed to high level in immune cells, On the plasma membrane it assembles with P2X7A to form a heterotrimeric receptor that is much more stable than the typical P2X7A homotrimer. Quite intriguingly, the P2X7A/P2X7B heterotrimer has a much higher Ca permeability, pore-forming and growth-supporting activity. Our data suggest that P2X7B is a major modulator of P2X7A functions.
In summary, the studies on P2X7 receptors have clarified the role of the P2X7 receptor, extracellular ATP and adenosine in osteoclast fusion which is a fundamental step forward in the understanding of the role of purinergic signalling in osteoclast physiology and in bone homeostasis.
As our studies have also shown that intact P2X7 receptor function is important for maintaining a healthy skeleton, we have identified a number of genetic variations (single nucleotide polymorphisms) in the human P2X7 receptor and as we have shown an association between these and bone mass, bone loss and fracture risk in human subjects, it was relevant to demonstrate the isolated effects on bone cells of these polymorphisms. Based on the results from the cohort studies described below we selected a number of individuals from the Danish osteoporosis prevention cohort carrying the relevant genotypes for the P2X7 polymorphisms. From these individuals we obtained bone cells which we investigated the effect of the different genetic variations. Interestingly, we found that genetically based defects in the receptor function was also affecting the function of the bone cells, supporting the genetic association studies showing that individuals carrying certain genotypes had increased risk of osteoporosis and subsequently fragility fractures.
In vivo role of P2 receptors on normal bone physiology, response to mechanical stimulation and oestrogen deficiency
While the initial work primarily involved laboratory studies exploring P2 signalling pathways, the later studies in the project addressed the in vivo functions of the receptors. The approach taken was to use mouse models, in which distinct receptors were not expressed, the so-called knockout mice. The basic in vivo functions on bone formation and homeostasis as well as on bone mass and structure were determined. Studies addressing the role in osteoporosis-related bone loss were performed using ovariectomised mice, which is a widely established model of postmenopausal osteoporosis. Finally, the anabolic potential of the purinergic system was elucidated by performing studies on the involvement of the P2 receptors in mechanotransduction by applying mechanical loading to bones of the different relevant knockout strains compared to animal where the receptor was expressed normally. A number of knockout models were used including P2Y6, P2Y13, P2Y1, P2Y2, and P2X7. An extensive amount of work and investigations were conducted, including measurement of bone mass, serum markers of bone formation and -resorption, micro-CT scan of bone micro-structure, bone histology and -histomorphometry. Both young and older animals were investigated as the receptors might have differential effects at immature and mature skeletons.
Interestingly, all strains of P2-deficient mice studied have displayed a bone phenotype and each of these phenotypes is unique. These studies emphasise thus the importance of nucleotide signalling in the control of bone metabolism and the unique role of each receptor. Studies of the bone phenotype of P2X7 knockout mice performed before the ATPBONE project have provided conflicting results between two distinct models generated at Pfizer and GSK respectively. Mice generated at Pfizer exhibited a decreased bone mass due to decreased bone formation and increased bone resorption. The GSK mice had a mild phenotype with an increased cortical thickness. This apparent contradiction can probably be explained by the expression of a functional splice variant of P2X7 in the GSK mice. But these mice were generated in the B6 background which harbors a natural mutation of the P2X7 gene. We therefore developed a P2X7 knockout model in the BALB/cJ background and found an increased bone mass and decreased resorption in P2X7 knockout. In contrast in the P2Y1 knockout mice we found reduced trabecular bone mass in these animals, but the study was only conducted on long bones of dead animals and further studies are warranted to determine the exact roles of the P2Y1 receptor in the regulation of bone turnover in vivo.
The P2Y2 receptor is thought to mediate the inhibitory effects of ATP and UTP on bone mineralisation in vitro. Consistent with these observations, we found that trabecular and cortical bone mass was increased in the P2Y2 knockout mice; thus, deletion of the P2Y2 receptor could potentially limit the negative actions of extracellular nucleotides on bone. However, these studies are currently ongoing and all data are not analysed yet.
Preliminary results indicate that P2Y6 knockout mice have an increased bone mass that can be explained by the decreased resorptive function of osteoclasts. Bone resorption was also decreased in P2Y13 knockout mice, but this was explained by a decrease in the osteoclast number rather than osteoclast function. In these mice, decreased bone resorption was associated with decreased bone formation: this decreased bone turnover resulted in opposite effects on trabecular (decreased volume) and cortical (increased thickness) bone. A P2Y13 antagonist might thus be beneficial in osteoporosis which is characterised by an increased bone turnover.
These studies of knockout mice demonstrate thus that not less than five P2 receptors play a role in bone formation and / or resorption. This confirms the prediction that nucleotides acting via both P2X and P2Y receptors could play important roles in the regulation of bone metabolism. These important roles can be related to the fact that mechanical stimulation, which is an important factor in bone remodelling, stimulates the release of nucleotides. This was also supported by the finding that at least some of the receptors are involved in the anabolic responses to mechanical stimulation / physical exercise on bones, as well as in the regulation of the response to oestrogen withdrawal. Due to the labour-intensive nature of these studies, the work is still in progress and is expected to be completed during 2011 with the publication of the scientific articles as soon as possible thereafter.
Translational studies: P2 receptors in human osteoporosis aetiology and pathophysiology
During the second period of the ATPBONE project, some of the main objectives were to explore the role of aberrations in P2 receptor function in osteoporosis aetiology. As mentioned above the genetic defects in the P2 receptor genes were shown to affect normal receptor function and activity of bone cells in vitro, and we therefore tested the association of genetic polymorphisms with osteoporosis and fracture susceptibility in a number of cohorts in Denmark, UK and the Netherlands, which all are countries with high incidences of osteoporosis. Different target cohorts have been included, including healthy post-menopausal women, fracture patients, older men etc. Thus the associations have been determined in an extensive set of cohorts with different disease frequencies in order to get a full picture of etiological associations.
We have previously within the consortium demonstrated that polymorphisms in the P2X7 receptor are associated with both incidence of vertebral fractures and increased bone loss after menopause in women. The P2X7 receptor is highly polymorphic with more the 200 polymorphisms published. A number of these have been investigated in terms of effects on receptor function in haematological cells, which together with the results from our previous study resulted in that we decided to genotype for further P2X7 polymorphisms. We found that specific P2X7 receptor polymorphisms that are shown to have total loss-of-function effects on receptor function were associated with increased rate of bone loss in the cohorts studied and in some cohorts also with increased fracture incidence. Also polymorphisms in other P2 receptors were investigated, and the data analysis is currently ongoing.
Translational studies: Mechanical and pharmacological interventions in osteoporosis
The final part of the ATPBONE project aimed at integrating all the findings from the basic studies, the in vivo studies and some of the information from the genetic association studies. These studies intended to provide proof-of-concept of the anabolic potential of pharmacological modulation of purinergic signalling in osteoporosis treatment. In an mouse model of osteoporosis, a surgically induced menopause was created by removing the ovaries. This results in the loss of oestrogen stimulation and subsequently accelerated bone loss in the mice. Then the mice were treated with agonists and antagonists specific to certain P2 receptors for one month and the effect of the pharmacological treatment on the bone loss was assessed. The study conducted as a randomised, dose-response study, where data collection and treatment was blinded to minimise bias. The different treated groups were related to baseline controls, vehicle controls and sham operated animals. The Danish Animal Welfare Council had approved all animal procedures in advance (protocol: 2010/561-1795). As control treatment, parathyroid hormone was used as it is the most potent anabolic agent available for treatment of osteoporosis in the clinic. For efficacy measurements, the following materials / parameters were collected: Serum was collected for bone marker determination; femur, tibia, and spine were collected for micro-CT scan for determination of bone micro-structure, histomorphometry and histology, as well as bone strength measurements. Bone mass measurement were also performed. Four different receptors were targeted and results were highly encouraging. However, data analysis is still ongoing and is expected to be completed during 2011. The main results confirmed the P2 purinergic signalling system as a potential target for pharmacological treatment of bone loss and osteoporosis after oestrogen withdrawal.
Another important objective of this final period of the project was to develop and test a human model of mechanical loading which can be utilised to assess responses of purinergic markers to mechanical stimulation in vivo. The study has been carried out and seven healthy subject have successfully completed the experiments. Data analysis is currently ongoing.
The final objective in the interventional part of the project aimed at evaluating effects of a purinergic intervention on markers of bone metabolism in human subjects. This was a small-scale pilot study to investigate the effect of ATP infusion on bone markers of human osteoporotic subjects. As a human intervention study, this study needed ethical approval in order to be carried out. The legal requests for ethical permission for clinical trials with medication in the Netherlands have changed drastically during the ATPBONE project, especially with respect to the requirements of the preparation of ATP solutions for intravenous infusion. In the initial phase of ATPBONE (2008), the Dutch national legislation was adapted to comply with new EU regulations. This change in national legislation for ethical approval comprises the compulsory submission of an extensive investigational medicinal product dossier (IMPD) regarding the compound ATP, its safety for application in humans, and details as well as validation of the preparation of ATP solutions for intravenous infusion. Since ATP is prepared as an aseptic infusion solution, the IMPD must include a detailed protocol of the preparation process. At the moment of writing, no hospital pharmacy in southern Netherlands has the GMP facilities and permit required for preparation of aseptic infusion solutions for research purposes. For this reason, so far, we have not been able to obtain ethical permission for the planned pilot pharmacological intervention with ATP.
Conclusion
The studies performed during the ATPBONE project period has provided evidence for important roles for ATP as a signalling molecule in bone and for purinergic signalling in the regulation of bone formation and homeostasis as well as in the regulation of bone resorption. The initial part of the project provided detailed mechanistic knowledge of the individual P2 receptors in specific functions of bone cell activity, proliferation, differentiation as well as in cell-cell signalling, bone matrix formation and mineralisation. Also information on the metabolism of ATP has been provided through the project. In addition within the framework of the project, a new an important tool has been created in the form of the pmeLUC transgenic mouse that will prove invaluable for the study of ATP in not only bone but also other areas of health related research.
Based on the in vitro findings, in vivo studies were conducted showing important roles for P2 receptors and purinergic signalling in normal bone physiology, as well as in mechanotransduction. The different bone phenotypes in the individual knockout model investigated suggests that each receptor subtype have distinct roles in regulation of bone formation and resorption. This knowledge may prove important when targeting P2 receptors as new pharmacological targets in the treatment of osteoporosis. This potential was proven in the proof-of-concept studies where different P2 receptors were targeted in the in vivo osteoporosis model, where bone loss could be prevented by using receptor specific agonists and antagonists for certain receptors. Though further studies are warranted in order to pinpoint the exact receptors to target, the results obtained so far have definitely proven the potential impact this novel treatment modality may have.
In conclusion, the ATPBONE project has provided clear evidence that P2 purinergic signaling has important, yet complex roles in the regulation of bone turnover, and that it is a potential novel target for pharmacological treatment of osteoporosis.
Potential impact:
The vision
The overall aim of the ATPBONE project was to demonstrate the role of ATP and purinergic receptor signalling in the control of bone formation and homeostasis, thereby proving the potential of the system as a target for the development of new anabolic treatment regimens for osteoporosis. It is important that the results evolving from the project will ultimately reach the EU citizens. We have therefore taken steps to ensure that results are used for enforcing the impacts for the benefit of the patients in terms of significantly improved quality-of-life, for the benefit of the European countries and health-care systems in terms of reduced costs for treatment of osteoporosis-related fractures, for the benefit of European industry in terms of income-making related to drug development and sales, as well as for the benefit of the scientific community in terms of increased knowledge about bone biology in general and osteoporosis aetiology and pathogenesis in particular.
The task
Osteoporosis is a disease highly prevalent in the elderly population. Fractures related to osteoporosis are affecting quality-of-life of the patients and is a significant economical burden to society. As the number and proportion of elderly people is steadily increasing in most European countries and the rest of the World, the total societal costs of osteoporosis (including health insurance costs, losses in terms of human capital, and personal costs) as well the societal impact of functional disability and loss of quality of life of European citizens will increase dramatically over the next decades. It is estimated that the incidence of 1.7 million hip fractures in 1990 will increase to 6.3 million per year around 2050. In order to effectively prevent osteoporotic fractures, the disease should be diagnosed at an early stage, before the fractures appear. This is currently possible using bone mineral density scans. The second step after early diagnosis will be the early initiation of treatment in order to reduce fracture risk. For this purpose, the availability of effective and financially affordable treatment options will be essential. At present, a number of anti-resorptive drugs are available, but only one anabolic drug (teriparatide = PTH(1-34)) is available that can restore the lost bone and thereby reverse the disease. Moreover, teriparatide is expensive and restricted to a small subgroup of patients with most severe osteoporosis, since its mode of administration is by subcutaneous injections and the treatment duration is limited to only 24 months because of concerns over possible long-term side effects. Thus, new anabolic treatments are urgently needed.
The impact
The proposed project specifically aimed at unravelling the precise mechanism of involvement of the ATP and purinergic signalling system in bone formation and homeostasis, and to demonstrate the potential of the system for the development of new treatment modalities directed against osteoporosis. The studies performed have given strong evidence that activation of the system is one of the most potent activators of intracellular calcium, and of the potential of this system to modulate both bone formation and homeostasis in a concerted manner. Also pre-clinical studies have confirmed the strong potential of pharmacological intervention in the purinergic system as a powerful tool, since it is generally accepted that the optimal treatment of bone loss should be aimed at simultaneously stimulating bone formation and inhibiting bone resorption, where both these activities can be modulated through the purinergic system. Therefore, the ATPBONE project, as a joint effort combining the expertise of different leading groups in Europe and the world in this area, has succeeded in attaining the expected impacts. Most importantly, it has provided a coherent picture of the way in which the results can be pursued further to develop new anabolic agents for the treatment of osteoporosis. This may lead to more effective treatment options for patients, and thereby to delay in the onset of osteoporosis and thus reduction in fracture risk. The results from the project may also lead to more convenient ways of administration of purinergic pharmacologics, resulting in a significant increase in the quality-of-life for the patients. However the impacts reach far beyond the patients, as also the countries of the EU as well as most other countries in the world will benefit economically from improved treatment options for osteoporosis. The costs of treating osteoporosis-related fractures world-wide are immense. In the EU alone, the annual combined costs of all osteoporosis-related fractures is estimated to be approximately EUR 25 billion. Thus, developing new and widely available anabolic agents to treat osteoporosis will have enormous impact on the Member States and health systems around Europe, as well as in the rest of the world, and would be of huge economical importance to the society.
Moreover, the economical impact will not be restricted to the public sector or health care systems in the Member States. Since the European consortium collaborating in this project comprises most of the world's scientific leaders in this area, its results will have impact on the European biotech industry, since scientific success in the proposed project will be pursued further to develop agents that can be used in the clinical setting for modifying the system. This will involve small-to-medium sized European enterprises (biotech companies) that will collaborate on taking the findings of the project to further preclinical and clinical development. It is therefore of utmost importance to secure proprietary rights in every possible case.
The outcomes of the project will not only have impacts related to the identification of a possible new treatment target for osteoporosis, but the genetic analyses and the in vitro studies of the effect of genetic variations in bone cells have given significant insight into the pathogenesis and aetiology of osteoporosis. Further consolidating this knowledge about the genetic variations in the purinergic receptors and the association with osteoporosis may be used to identify individuals with a high propensity to develop osteoporosis. This means that identifying these patients at an early stage could lead to early treatment of the disease in such high risk patients. This would further impact the quality-of-life of the patients as well as the costs for fracture treatment in the health care system.
Other impacts evolving from the project are an increased awareness of osteoporosis in the public, as the dissemination measures taken by the consortium involves both press releases, contact with patient organisations, and a website addressing different aspects of osteoporosis and bone biology. Patients will also be aware of the many efforts taken to improve therapeutic approaches of the disease. Moreover, the level of awareness in politicians and decision makers of the epidemic size of the osteoporosis problem has and will also increase. This would be of major importance to influence the decision-making and allocation of health care resources to measures aimed at improving osteoporosis diagnosis and treatment. Thus a self-enforcing process has presumably been initiated by the project contributing to the wellness of European citizens and the society.
Steps needed to bring about the impact
The ATPBONE project was designed to take a major step forward in elucidating essential aspects of the P2 receptor signalling mechanisms involved in the regulation of bone formation and homeostasis, within the relatively limited time of the project. Thus, all aspects of the signalling system have been addressed. The project started with a basic characterisation of the signalling molecule (ATP) with respect to release mechanisms and stimuli, degradation and conversion. Next the expression pattern as well as the in vitro functions of the P2 receptors were addressed, to fully elucidate their role in osteoblast activity (matrix formation and mineralisation), as well as in osteoclast formation and resorptive activity in order to characterise bone homeostasis. Also the intracellular pathways activated by the receptor were investigated. To test the physiological significance and role of the signalling system, a number of genetically modified mouse models of osteoporosis were examined. Based on the findings from the laboratory and mouse studies, pre-clinical studies were performed demonstrating the usefulness of the system to modify bone formation and homeostasis in vivo by treatment with pharmacological agents active at the receptors.
As indicated above, the work plan of this project addressed all aspects necessary to fully elucidate the role of purinergic signalling in bone, as well as the potential of the purinergic signalling system in the treatment of osteoporosis in vivo. However, it was outside the scope and budget constraints to conduct clinical studies in humans as clinical trials require significantly more resources than available at the moment.
As evidenced by the number of different studies involved in the elucidation of signalling pathways, it was impossible to perform the current project in only one country. It was therefore of utmost importance to perform the project as a European collaborative project, not only to create the synergy between the participating scientific groups, but also to increase the awareness and impact in the whole EU instead of regionally or nationally.
In summary, the results from the ATPBONE project has the potential:
1. To contribute to an improved health and quality of life of the European citizens, osteoporosis patients especially, by delaying or preventing osteoporosis-related fractures.
2. To have a favourable impact on the economy of the community and healthcare systems, by affecting the competitiveness of the health-related industries or businesses especially small and medium-sized entreprises (SMEs) in the EU.
Dissemination and exploitation of project results
The results from the project are potentially of benefit to the public by providing new knowledge that may lead to new anabolic treatment strategies for osteoporosis, and therefore the dissemination of these results is of interest to the European citizens. The citizens of Europe have financed the project through taxes, and therefore have the right to be informed of the results and how the money is spent. The consortium has therefore taken measures to secure that the results from the project are widely disseminated to the public and to the scientific community. These measures include, but are not limited to the following:
- a website: http://www.atpbone.eu
- press releases describing important progress and findings in the project;
- contact with national osteoporosis patient organisations for articles in the membership magazines, and oral presentations at meetings in these societies.
The consortium has also taken measures to disseminate the results from the project to the scientific world, so that other scientists can use the acquired knowledge. This has taken place by:
- publication of the results in high rank international peer-reviewed scientific journals;
- oral and / or poster presentations at international scientific meetings and conventions
Thus the results from the project have been and will be disseminated to both the public, so that the European citizens will be informed about recent advances in osteoporosis treatment and aetiology as well as about the use of their tax money. Dissemination of the results to the scientific world will secure that the results can be used in the widest possible way to increase the knowledge about osteoporosis and bone biology, and to increase the chances that the findings will lead to improvement of treatment and handling of osteoporosis in the future.
Dissemination activities to scientific community:
During the project period, the ATPBONE consortium members have taken a number of measures to disseminate the knowledge emerging from the project. Naturally, there is a strong focus of disseminating the results from the scientific experiments to the academic world and scientific community in order to spread the highly interesting findings so that other can also build new experiments and knowledge on the data. Until now, 26 articles have been published already three years after the initiation of the project. Data analysis for most of the studies performed is still ongoing and it is estimated that an additional 50 peer reviewed scientific papers will be published within the next two years after the conclusion of the project period. Also, the scientists in the consortium have also been highly active in disseminating the results at national and international scientific meetings. A large number of abstracts have been submitted to these meeting, and many more are expected. Several of the abstracts have been selected for oral presentations and some of these have even been awarded prizes and awards. The focus of the ATPBONE project has generated significant interest within the scientific community. First, the consortium was asked to provide a number of review articles to a special issue of Frontiers in Bioscience, where also other leading non-European experts in the field of purinergic signalling in bone were invited to participate. Furthermore, based on the results of the ATPBONE project, the European Calcified Tissue Society has decided to have a special session on purinergic signalling in bone at the next annual international meeting in Stockholm in May 2012 with two of the ATPBONE members as keynote speakers. Thus, even with a lot of data still remaining to present from the project there has been a high interest from the scientific community.
Within the consortium we have taken a special step to increase the exchange of knowledge between groups but also to promote the ATPBONE project in the scientific community. Every six months, we have arranged an ATPBONE symposium. This symposium has moved around among the seven participating institutions, and has included presentations both by the principal investigators, young scientists from the participating centres as well as invited international capacities within the bone field. These symposia have received much interest from also outside the consortium and have been well attended. Furthermore, it is planned to continue these symposia also after the conclusion of the project, where annual meetings are planned for the exchange of ideas and for the continued collaboration among the partners. The ATPBONE symposia have also provided grounds for building a strong scientific network throughout the EU. Both senior and younger scientists have participated in the meeting, and important social activities have also been part of the meetings. This has increased the contact between both older and younger scientist with the added benefit of strengthening the network for the future of these scientists. This has been important and has also consolidated the future collaboration within the group and among all involved scientists.
Dissemination activities to target groups:
However, as it is important to promote and disseminate the findings also to other groups, the consortium has taken a number of measures to also disseminate the existence and findings from the project to other target groups, such as policy makers and the civil society. First, we have invested a great deal of time and human resources in promoting both the project as well as the knowledge of the European Commission (EC) and FP7 to other researchers, policy makers and other relevant persons. The coordinating center has engaged in a collaboration with the Danish contact point, Eurocenter, to teach at courses and information meetings for the scientific community and the industry to promote FP7. We have also been active in disseminating the knowledge of the project to the general public and to patients. During the course of the project, there have been several articles and interviews focusing on the project in patient magazines, magazines for employees of the health care system etc. In addition, we have one major article planned for publication in May in the magazine of the Danish Osteoporosis Organization (patient organisation), and another has already been published in the magazine of the Dutch Osteoporosis Organization. In addition, all consortium partners are focused on contacting the national osteoporosis organisations to have similar publications describing the ATPBONE project so that all relevant patients acquire knowledge about the project and the possible future implications.
Other measures have also been taken to spread the knowledge of the project. These have included oral presentations in patient organisations, participation in and presentation of the projects at science fairs (University of Ferrara) for adolescents, where the work package leader was awarded the best project presented.
Exploitation
The results from the project can potentially generate proprietary rights that need to be secured by patent applications. Throughout the duration of the project the consortium has constantly focused on whether there were patentable ideas and results that need to be secured. Until now, due to the relatively basic nature of the project, there have not been any patentable findings yet. However, the results obtained are highly interesting and further studies are planned to support potential patent applications and further exploitation of the foreground. As soon as it is relevant and the necessary amount of supporting data becomes available the participants in the consortium will take steps to determine whether a patent application is appropriate. The collaborators will also decide which participants have been involved in the patentable ideas / results.
List of websites:
For more information on the project and the consortium, please see the ATPBONE webpage at http://www.ATPBone.eu or contact scientific coordinator Niklas Rye Jørgensen (niklas@dadlnet.dk via e-mail).
Purinergic signalling, where ATP and its metabolite adenosine act as extracellular signalling molecules is a rapidly expanding field. Evidence has accumulated that the purinergic system plays a central role in bone physiology. Thus, the overall aim of the present project is to provide mechanistic basis for nucleotide-based therapeutics in osteoporosis. For this purpose, we have gathered in the ATPBONE consortium the main research groups in the European Union (EU) working in the field of osteoporosis and purinergic signalling. Participating institutions are Copenhagen University Hospital Glostrup, Denmark; University College London, United Kingdom (UK); University of Maastricht, the Netherlands; University of Ferrara, Italy; University of Liverpool, UK; University of Sheffield, UK; and Université Libre de Bruxelles, Belgium. The project was funded with EUR 3 million through the EU Seventh Framework Programme (FP7), and commenced 1 January 2008 with a duration of 3 years.
The work plan included:
(A) basic in vitro investigations on ATP release and signalling;
(B) a translational section involving in vivo experiments including transgenic mice,;and
(C) studies of human cohorts and clinical intervention studies.
Thus, the project involved on the one hand a fundamental part, aiming to give a basic description of the purinergic signalling system, and on the other hand a translational part to demonstrate the importance of the purinergic system in osteoporosis treatment. Multiple experimental approaches have been made, including physiological challenge of bone metabolism by mechanical loading, and pharmacological intervention on the purinergic signalling system.
The results have been highly interesting and encouraging. In the consortium, we have obtained a very detailed insight into the basic mechanisms of purinergic signalling in bone, as well as a better understanding of the regulation of bone metabolism including the formation of new bone, degradation of old bone tissue as well as the mineralisation of bone matrix. The in vivo experiments have given us important new knowledge of the roles of the P2 purinergic receptors on the regulation on bone turnover in the living skeleton, and in the translational studies we have provided new and significant evidence that genetic variations in the genes coding for the P2 receptors are associated with increased bone loss, osteoporosis and fragility fractures, indicating an important role of P2 receptor defects in osteoporosis pathophysiology. Finally, the highly encouraging early pre-clinical studies in mice have provided evidence for the P2 receptors as pharmaceutical targets for anti-osteoporotic treatments, which will be pursued further within the frames of the consortium even after the project is officially closed.
Thus, the project has provided both mechanistic insight into basic mechanisms of bone turnover as well as into osteoporosis pathophysiology, and the role and importance of the P2 receptors in bone metabolic diseases will be further exploited in future projects within the consortium.
For more information on the project and the consortium, please see the ATPBONE webpage at http://www.ATPBone.eu or contact scientific coordinator Niklas Rye Jørgensen (niklas@dadlnet.dk via mail).
Project context and objectives:
Osteoporosis is a widespread systemic disorder characterised by decreased bone mass and deterioration of bone micro-architecture. The social economic burden is high as it is estimated that in Europe about 1 in 3 women and 1 in 8 men above the age of 50 will suffer an osteoporotic fracture. These numbers are expected to increase exponentially worldwide, as the ageing population increases significantly and the sedentary lifestyle and changes in nutritional habits are making the impact even larger. Osteoporosis results from a combination of genetic, environmental and other risk factors that affect both peak bone mass and the rate of bone loss. These factors include gender, age, age at menopause, use of glucocorticoids, chronic disease, diet, race, lifestyle and physical activity. Osteoporosis may be either primary or secondary. Bone loss is a result of a dysbalance of bone formation and bone resorption. In postmenopausal osteoporosis, which is the most common form, there is evidence that bone resorption is increased without sufficient compensatory increase in bone formation. Effective anti-resorptive therapy is available to reduce the risk of osteoporotic fractures: Calcium and vitamin D, selective estrogen-receptor modulators, bisphosphonates and strontium ranelate. However, even though these agents reduce bone resorption, they have little, if any, effect on bone formation. Parathyroid hormone (PTH) treatment is anabolic and may be used in very severe cases of osteoporosis for up to one an a half year in European countries, leading to increased bone quality and strength. However, PTH is currently the only anabolic agent for osteoporosis treatment and with a limited treatment period of use due to the possibility of stimulating osteosarcoma in patients. In view of the high disease load of osteoporosis and its expected dramatic rise over the next decades, as well as the very limited accessibility to anabolic treatments it is pressing to create new effective pharmaceuticals for fighting osteoporosis in the future. For this purpose, it is of major importance to elucidate the currently ill-understood basic mechanisms of bone metabolism and function on a cellular level particularly receptor signalling and pathways involved in the normal osteoclast and osteoblast function.
Purinergic signalling, where ATP and its metabolite adenosine act as extracellular signalling molecules in a whole range of tissues, is a rapidly expanding field. Over the past decade, evidence has accumulated that the purinergic system plays a central role in bone physiology. Osteoblast and osteoclast physiology is finely tuned by extracellular nucleotides, and so are bone deposition and resorption. Signalling by extracellular ATP in bone is even more fascinating due to the exquisite mechanical properties of this tissue, and to the known ATP-releasing ability of mechanical stresses and tensions, to the point that it is now widely believed that release of ATP from bone cells is one of the main transduction pathways of mechanical stimulation. ATP has also been recognised as one of the most important early signalling molecules released by injured cells during the early phases of inflammation also known as danger associated molecular patterns (DAMPs), while adenosine has an well-established role as a potent immunosuppresive factor. Like most immuno-modulating agents, ATP can act either as an immunosuppressive or an immunostimulatory factor, depending on the dose, the cytokine microenviroment and the pattern of P2 receptor expression of the target cells. This may be particularly relevant in the bone microenvironment where nucleotides may modulate expression of membrane or soluble receptors involved in osteoblast/osteoclast maturation, such as receptor activator of nuclear factor kappa (RANK) and its ligand (RANKL) or osteoprotegerin, and may enhance sensitivity of bone cells to hormones such as PTH.
The overall aim of the proposal, based on current knowledge of the purinergic signalling system and our previous work in the field, is to provide mechanistic basis for nucleotide-based therapeutics in osteoporosis, effective through modulation of bone formation and homeostasis. The large number of different P2 receptor subtypes with widely differing affinity for extracellular nucleotides and the many different biologic responses that can be elicited by stimulating each of them, makes the 'purinergic system' a highly flexible target for the design of innovative molecules for the treatment of bone disease.
Project results:
Overall strategy of work plan
The proposed project was designed as a multidisciplinary approach to elucidate the role of ATP signalling through P2 receptors on bone with special emphasis on the effects on bone formation and homeostasis. The work plan included:
A) fundamental in vitro investigations on ATP release and signalling;
B) a translational section using animal experiments including transgenic mice; and
C) studies of human cohorts and clinical intervention studies.
Thus the ATPBONE project aimed at giving both a basic description of the purinergic signalling system and a translational part demonstrating the value of the system in osteoporosis treatment in a highly interrelated and integrated project. Interventions included both mechanical loading, as the normal physiologic stimulus for bone formation, and pharmacological intervention in the purinergic signalling system. Potential interactions of the purinergic signalling system with other established and novel factors affecting bone metabolism, such as oestrogen, parathyroid hormone, prostaglandins, and mediators of inflammation and oxidative stress, were examined.
ATP as a signalling molecule
The objective of the first part of the project was to describe the signalling molecule, ATP, in terms of demonstrating:
a) its mechanisms of release from bone cells; and
b) the stimuli leading to enhancement of release.
Particular emphasis was paid to the effects of mechanical stimulation, since this is an important stimulus of bone growth. The mechanically induced ATP release was tested both in vitro and in vivo partially using a new transgenic mouse created within the consortium. This mouse expresses plasma membrane-bound recombinant luciferase (pmeLUC) in order to visualise in vivo ATP release. The effect of other physiologically relevant stimuli such as hydroxyapatite and hypoxia were be tested. Since nucleotides are rapidly degraded by enzymes and the effect of nucleotides on bone cells is therefore influenced by the degradation and conversion patterns, the initial work also addressed the mechanisms for degradation and conversion of nucleotides.
During the work on this objective, we demonstrated that bone forming cells (osteoblasts) in cultures release ATP constitutively. Mechanical stimulation in the form of fluid displacement stimulates the release of ATP from osteoblasts, which was confirmed in three different model systems. Three types of mechanical loading have been applied, fluid displacement, shaking and vibration. Results have demonstrated that fluid displacement, which generates fluid shear forces, consistently enhances the release of ATP into conditioned medium and is a better stimulus than shaking or vibration. This indicates that fluid flow might be the most physiological stimulus in vivo. Also the substrate on which the cells are attached influences the release of ATP, and the response to fluid shear. Cells cultured on calcified substrates tend to release more ATP than cells on plastic culture plates. These studies have also revealed that ATP can bind to mineralised matrix. Also the bone resorbing cells, osteoclasts, were shown to be able to release ATP when stimulated mechanically.
To investigate whether ATP is released from bone in vivo, we engineered a mouse expressing a reporter for ATP release, also called pmeLUC. Examining this mouse in a whole body luminometer enables the detection of ATP release in living animals. Generation of this transgenic animal has requested a considerable effort, that however was well spent as the pmeLUC mouse appears to be a very sensitive detector of ATP release into the extracellular space, and therefore of mechanical stimulation, tissue damage or inflammation. Along the same lines, we have further developed the HEK293-pmeLUC biosensor that was at the basis of the construction of the pmeLUC-mouse. We can now safely conclude that the HEK293-pmeLUC biosensor is a very sensitive and reliable indicator of changes in the extracellular ATP concentration in a number of different conditions ranging from mechanical stimulation to cancer, from graft-versus-host to contact sensitivity reactions. Lack of a reliable sensor for the measurement of extracellular ATP has long been a major drawback in the purinergic receptor field, and the engineering of the pmeLUC probe is a real break-through. One of the main objectives was to determine whether ATP is release in bone upon mechanical stimulation in vivo. We therefore applied a new model of bone loading. Traditional in vivo models of bone loading have relied on passive loading. We have developed a novel method which relies on muscle activation. Stimulator devices are implanted in the animals stimulating the nerve to one hindlimb, causing muscular contraction and subsequent loading of bones. By using this model we have demonstrated release of ATP in vivo in response to loading. Furthermore, we have shown that mechanical loading induces changes in expression of multiple genes in muscle and in bone.
To further study which factors other than mechanical forces that can induce ATP release in bone cells, we examined a range of stimuli that are known to affect nucleotide release from other cell types. We found that low oxygen tension (hypoxia) as well as other types of mechanical stimulation could induce ATP release from the bone cells.
The last part of the studies on ATP focused on the regulation of ATP concentrations outside the cells. Once released the activity of nucleotides are limited by the actions of enzymes which convet and degrade nucleotides to their corresponding nucleoside and phosphate. We have studied the expression of these enzymes on osteoblasts under normal and acidic conditions. We have shown that osteoblasts express multiple enzymes and that the expression of these enzymes changes during osteoblast differentiation. Work is ongoing to investigate other factors which regulate the expression and activity of these enzymes.
Expression and signalling pathways of P2 purinergic receptors
There are a number of different P2 purinergic receptors belonging to the two subgroups, P2Y and P2X, These are clearly distinct in nature and in order to understand the functions of the purinergic signalling system in bone, it is important to determine the expression of P2 receptors in bone cells as well as the effects of receptor activation on bone formation and degradation. In addition, hormones (oestrogen and parathyroid hormone) and other factors that regulate bone turnover, might interact with the P2 receptors' functions. Therefore, the next part of the project focused on investigating the exact expression, functions and interactions with hormones of the individual and distinct P2 receptors.
During the work on this objective, we described the detailed expression of P2 receptors on bone cells, including the on the bone forming osteoblast and on the bone degrading osteoclasts. We also showed that the receptors play significant roles in bone, modulating both osteoblast and osteoclast function and that the expression pattern changes during the differentiation of the cells. We also showed that stimulation or inhibition of the different receptors with universal or specific agonists and antagonists could modulate the activity of the bone cells so that bone forming activity could either be stimulated or inhibited depending on the specific receptor targeted. We also used cells from mice that did not express single specific receptors, confirming the findings from the agonist / antagonist studies.
As parathyroid hormone is the most important regulator of bone metabolism in vivo, it is highly relevant to investigate whether there is an interaction between parathyroid hormone signaling a dn purinergic signaling. We have previously shown that dual activation of P2 receptors and parathyroid hormone receptors on bone forming cells leads to a synergistic action. The studies demonstrated that transient exposure of bone cells to ATP sensitises the cells to the action of parathyroid hormone for up to nine hours. Furthermore, the parathyroid hormone and ATP also interacts on genes including that regulates the formation and activity of the bone resorbing osteoclasts. This synergy suggests a mechanism whereby locally released extracellular nucleotides can enhance the sensitivity of the cells to systemic hormones and thereby activate bone remodelling at target sites. We have extended these observations to other systemic hormones including the gut hormones, glucose-dependent insulinotropic polypeptide (GIP) and the adipocyte-derived hormone, adiponectin.
One of the subtypes of the P2 receptors has received special attention. It is the P2X7 receptor that has distinct features compared to the other P2X receptors as it can form a non-selective and large conductance pore. There has been a long-standing issue of the participation of pannexin-1 to the generation of the P2X7 pore. Our data provide unequivocal demonstration that pannexin-1 is not a constituent of the P2X7 pore, but we have found that pannexin-1 has a key role in ATP release, In addition we have performed an extensive investigation of the down-hill signalling molecules from the P2X7 receptor.
The P2X7 receptor is highly variable and we have therefore further investigated this. We have cloned and fully characterised a novel truncated splice variant of the human P2X7 receptor named P2X7B. This isoform is expressed to high level in immune cells, On the plasma membrane it assembles with P2X7A to form a heterotrimeric receptor that is much more stable than the typical P2X7A homotrimer. Quite intriguingly, the P2X7A/P2X7B heterotrimer has a much higher Ca permeability, pore-forming and growth-supporting activity. Our data suggest that P2X7B is a major modulator of P2X7A functions.
In summary, the studies on P2X7 receptors have clarified the role of the P2X7 receptor, extracellular ATP and adenosine in osteoclast fusion which is a fundamental step forward in the understanding of the role of purinergic signalling in osteoclast physiology and in bone homeostasis.
As our studies have also shown that intact P2X7 receptor function is important for maintaining a healthy skeleton, we have identified a number of genetic variations (single nucleotide polymorphisms) in the human P2X7 receptor and as we have shown an association between these and bone mass, bone loss and fracture risk in human subjects, it was relevant to demonstrate the isolated effects on bone cells of these polymorphisms. Based on the results from the cohort studies described below we selected a number of individuals from the Danish osteoporosis prevention cohort carrying the relevant genotypes for the P2X7 polymorphisms. From these individuals we obtained bone cells which we investigated the effect of the different genetic variations. Interestingly, we found that genetically based defects in the receptor function was also affecting the function of the bone cells, supporting the genetic association studies showing that individuals carrying certain genotypes had increased risk of osteoporosis and subsequently fragility fractures.
In vivo role of P2 receptors on normal bone physiology, response to mechanical stimulation and oestrogen deficiency
While the initial work primarily involved laboratory studies exploring P2 signalling pathways, the later studies in the project addressed the in vivo functions of the receptors. The approach taken was to use mouse models, in which distinct receptors were not expressed, the so-called knockout mice. The basic in vivo functions on bone formation and homeostasis as well as on bone mass and structure were determined. Studies addressing the role in osteoporosis-related bone loss were performed using ovariectomised mice, which is a widely established model of postmenopausal osteoporosis. Finally, the anabolic potential of the purinergic system was elucidated by performing studies on the involvement of the P2 receptors in mechanotransduction by applying mechanical loading to bones of the different relevant knockout strains compared to animal where the receptor was expressed normally. A number of knockout models were used including P2Y6, P2Y13, P2Y1, P2Y2, and P2X7. An extensive amount of work and investigations were conducted, including measurement of bone mass, serum markers of bone formation and -resorption, micro-CT scan of bone micro-structure, bone histology and -histomorphometry. Both young and older animals were investigated as the receptors might have differential effects at immature and mature skeletons.
Interestingly, all strains of P2-deficient mice studied have displayed a bone phenotype and each of these phenotypes is unique. These studies emphasise thus the importance of nucleotide signalling in the control of bone metabolism and the unique role of each receptor. Studies of the bone phenotype of P2X7 knockout mice performed before the ATPBONE project have provided conflicting results between two distinct models generated at Pfizer and GSK respectively. Mice generated at Pfizer exhibited a decreased bone mass due to decreased bone formation and increased bone resorption. The GSK mice had a mild phenotype with an increased cortical thickness. This apparent contradiction can probably be explained by the expression of a functional splice variant of P2X7 in the GSK mice. But these mice were generated in the B6 background which harbors a natural mutation of the P2X7 gene. We therefore developed a P2X7 knockout model in the BALB/cJ background and found an increased bone mass and decreased resorption in P2X7 knockout. In contrast in the P2Y1 knockout mice we found reduced trabecular bone mass in these animals, but the study was only conducted on long bones of dead animals and further studies are warranted to determine the exact roles of the P2Y1 receptor in the regulation of bone turnover in vivo.
The P2Y2 receptor is thought to mediate the inhibitory effects of ATP and UTP on bone mineralisation in vitro. Consistent with these observations, we found that trabecular and cortical bone mass was increased in the P2Y2 knockout mice; thus, deletion of the P2Y2 receptor could potentially limit the negative actions of extracellular nucleotides on bone. However, these studies are currently ongoing and all data are not analysed yet.
Preliminary results indicate that P2Y6 knockout mice have an increased bone mass that can be explained by the decreased resorptive function of osteoclasts. Bone resorption was also decreased in P2Y13 knockout mice, but this was explained by a decrease in the osteoclast number rather than osteoclast function. In these mice, decreased bone resorption was associated with decreased bone formation: this decreased bone turnover resulted in opposite effects on trabecular (decreased volume) and cortical (increased thickness) bone. A P2Y13 antagonist might thus be beneficial in osteoporosis which is characterised by an increased bone turnover.
These studies of knockout mice demonstrate thus that not less than five P2 receptors play a role in bone formation and / or resorption. This confirms the prediction that nucleotides acting via both P2X and P2Y receptors could play important roles in the regulation of bone metabolism. These important roles can be related to the fact that mechanical stimulation, which is an important factor in bone remodelling, stimulates the release of nucleotides. This was also supported by the finding that at least some of the receptors are involved in the anabolic responses to mechanical stimulation / physical exercise on bones, as well as in the regulation of the response to oestrogen withdrawal. Due to the labour-intensive nature of these studies, the work is still in progress and is expected to be completed during 2011 with the publication of the scientific articles as soon as possible thereafter.
Translational studies: P2 receptors in human osteoporosis aetiology and pathophysiology
During the second period of the ATPBONE project, some of the main objectives were to explore the role of aberrations in P2 receptor function in osteoporosis aetiology. As mentioned above the genetic defects in the P2 receptor genes were shown to affect normal receptor function and activity of bone cells in vitro, and we therefore tested the association of genetic polymorphisms with osteoporosis and fracture susceptibility in a number of cohorts in Denmark, UK and the Netherlands, which all are countries with high incidences of osteoporosis. Different target cohorts have been included, including healthy post-menopausal women, fracture patients, older men etc. Thus the associations have been determined in an extensive set of cohorts with different disease frequencies in order to get a full picture of etiological associations.
We have previously within the consortium demonstrated that polymorphisms in the P2X7 receptor are associated with both incidence of vertebral fractures and increased bone loss after menopause in women. The P2X7 receptor is highly polymorphic with more the 200 polymorphisms published. A number of these have been investigated in terms of effects on receptor function in haematological cells, which together with the results from our previous study resulted in that we decided to genotype for further P2X7 polymorphisms. We found that specific P2X7 receptor polymorphisms that are shown to have total loss-of-function effects on receptor function were associated with increased rate of bone loss in the cohorts studied and in some cohorts also with increased fracture incidence. Also polymorphisms in other P2 receptors were investigated, and the data analysis is currently ongoing.
Translational studies: Mechanical and pharmacological interventions in osteoporosis
The final part of the ATPBONE project aimed at integrating all the findings from the basic studies, the in vivo studies and some of the information from the genetic association studies. These studies intended to provide proof-of-concept of the anabolic potential of pharmacological modulation of purinergic signalling in osteoporosis treatment. In an mouse model of osteoporosis, a surgically induced menopause was created by removing the ovaries. This results in the loss of oestrogen stimulation and subsequently accelerated bone loss in the mice. Then the mice were treated with agonists and antagonists specific to certain P2 receptors for one month and the effect of the pharmacological treatment on the bone loss was assessed. The study conducted as a randomised, dose-response study, where data collection and treatment was blinded to minimise bias. The different treated groups were related to baseline controls, vehicle controls and sham operated animals. The Danish Animal Welfare Council had approved all animal procedures in advance (protocol: 2010/561-1795). As control treatment, parathyroid hormone was used as it is the most potent anabolic agent available for treatment of osteoporosis in the clinic. For efficacy measurements, the following materials / parameters were collected: Serum was collected for bone marker determination; femur, tibia, and spine were collected for micro-CT scan for determination of bone micro-structure, histomorphometry and histology, as well as bone strength measurements. Bone mass measurement were also performed. Four different receptors were targeted and results were highly encouraging. However, data analysis is still ongoing and is expected to be completed during 2011. The main results confirmed the P2 purinergic signalling system as a potential target for pharmacological treatment of bone loss and osteoporosis after oestrogen withdrawal.
Another important objective of this final period of the project was to develop and test a human model of mechanical loading which can be utilised to assess responses of purinergic markers to mechanical stimulation in vivo. The study has been carried out and seven healthy subject have successfully completed the experiments. Data analysis is currently ongoing.
The final objective in the interventional part of the project aimed at evaluating effects of a purinergic intervention on markers of bone metabolism in human subjects. This was a small-scale pilot study to investigate the effect of ATP infusion on bone markers of human osteoporotic subjects. As a human intervention study, this study needed ethical approval in order to be carried out. The legal requests for ethical permission for clinical trials with medication in the Netherlands have changed drastically during the ATPBONE project, especially with respect to the requirements of the preparation of ATP solutions for intravenous infusion. In the initial phase of ATPBONE (2008), the Dutch national legislation was adapted to comply with new EU regulations. This change in national legislation for ethical approval comprises the compulsory submission of an extensive investigational medicinal product dossier (IMPD) regarding the compound ATP, its safety for application in humans, and details as well as validation of the preparation of ATP solutions for intravenous infusion. Since ATP is prepared as an aseptic infusion solution, the IMPD must include a detailed protocol of the preparation process. At the moment of writing, no hospital pharmacy in southern Netherlands has the GMP facilities and permit required for preparation of aseptic infusion solutions for research purposes. For this reason, so far, we have not been able to obtain ethical permission for the planned pilot pharmacological intervention with ATP.
Conclusion
The studies performed during the ATPBONE project period has provided evidence for important roles for ATP as a signalling molecule in bone and for purinergic signalling in the regulation of bone formation and homeostasis as well as in the regulation of bone resorption. The initial part of the project provided detailed mechanistic knowledge of the individual P2 receptors in specific functions of bone cell activity, proliferation, differentiation as well as in cell-cell signalling, bone matrix formation and mineralisation. Also information on the metabolism of ATP has been provided through the project. In addition within the framework of the project, a new an important tool has been created in the form of the pmeLUC transgenic mouse that will prove invaluable for the study of ATP in not only bone but also other areas of health related research.
Based on the in vitro findings, in vivo studies were conducted showing important roles for P2 receptors and purinergic signalling in normal bone physiology, as well as in mechanotransduction. The different bone phenotypes in the individual knockout model investigated suggests that each receptor subtype have distinct roles in regulation of bone formation and resorption. This knowledge may prove important when targeting P2 receptors as new pharmacological targets in the treatment of osteoporosis. This potential was proven in the proof-of-concept studies where different P2 receptors were targeted in the in vivo osteoporosis model, where bone loss could be prevented by using receptor specific agonists and antagonists for certain receptors. Though further studies are warranted in order to pinpoint the exact receptors to target, the results obtained so far have definitely proven the potential impact this novel treatment modality may have.
In conclusion, the ATPBONE project has provided clear evidence that P2 purinergic signaling has important, yet complex roles in the regulation of bone turnover, and that it is a potential novel target for pharmacological treatment of osteoporosis.
Potential impact:
The vision
The overall aim of the ATPBONE project was to demonstrate the role of ATP and purinergic receptor signalling in the control of bone formation and homeostasis, thereby proving the potential of the system as a target for the development of new anabolic treatment regimens for osteoporosis. It is important that the results evolving from the project will ultimately reach the EU citizens. We have therefore taken steps to ensure that results are used for enforcing the impacts for the benefit of the patients in terms of significantly improved quality-of-life, for the benefit of the European countries and health-care systems in terms of reduced costs for treatment of osteoporosis-related fractures, for the benefit of European industry in terms of income-making related to drug development and sales, as well as for the benefit of the scientific community in terms of increased knowledge about bone biology in general and osteoporosis aetiology and pathogenesis in particular.
The task
Osteoporosis is a disease highly prevalent in the elderly population. Fractures related to osteoporosis are affecting quality-of-life of the patients and is a significant economical burden to society. As the number and proportion of elderly people is steadily increasing in most European countries and the rest of the World, the total societal costs of osteoporosis (including health insurance costs, losses in terms of human capital, and personal costs) as well the societal impact of functional disability and loss of quality of life of European citizens will increase dramatically over the next decades. It is estimated that the incidence of 1.7 million hip fractures in 1990 will increase to 6.3 million per year around 2050. In order to effectively prevent osteoporotic fractures, the disease should be diagnosed at an early stage, before the fractures appear. This is currently possible using bone mineral density scans. The second step after early diagnosis will be the early initiation of treatment in order to reduce fracture risk. For this purpose, the availability of effective and financially affordable treatment options will be essential. At present, a number of anti-resorptive drugs are available, but only one anabolic drug (teriparatide = PTH(1-34)) is available that can restore the lost bone and thereby reverse the disease. Moreover, teriparatide is expensive and restricted to a small subgroup of patients with most severe osteoporosis, since its mode of administration is by subcutaneous injections and the treatment duration is limited to only 24 months because of concerns over possible long-term side effects. Thus, new anabolic treatments are urgently needed.
The impact
The proposed project specifically aimed at unravelling the precise mechanism of involvement of the ATP and purinergic signalling system in bone formation and homeostasis, and to demonstrate the potential of the system for the development of new treatment modalities directed against osteoporosis. The studies performed have given strong evidence that activation of the system is one of the most potent activators of intracellular calcium, and of the potential of this system to modulate both bone formation and homeostasis in a concerted manner. Also pre-clinical studies have confirmed the strong potential of pharmacological intervention in the purinergic system as a powerful tool, since it is generally accepted that the optimal treatment of bone loss should be aimed at simultaneously stimulating bone formation and inhibiting bone resorption, where both these activities can be modulated through the purinergic system. Therefore, the ATPBONE project, as a joint effort combining the expertise of different leading groups in Europe and the world in this area, has succeeded in attaining the expected impacts. Most importantly, it has provided a coherent picture of the way in which the results can be pursued further to develop new anabolic agents for the treatment of osteoporosis. This may lead to more effective treatment options for patients, and thereby to delay in the onset of osteoporosis and thus reduction in fracture risk. The results from the project may also lead to more convenient ways of administration of purinergic pharmacologics, resulting in a significant increase in the quality-of-life for the patients. However the impacts reach far beyond the patients, as also the countries of the EU as well as most other countries in the world will benefit economically from improved treatment options for osteoporosis. The costs of treating osteoporosis-related fractures world-wide are immense. In the EU alone, the annual combined costs of all osteoporosis-related fractures is estimated to be approximately EUR 25 billion. Thus, developing new and widely available anabolic agents to treat osteoporosis will have enormous impact on the Member States and health systems around Europe, as well as in the rest of the world, and would be of huge economical importance to the society.
Moreover, the economical impact will not be restricted to the public sector or health care systems in the Member States. Since the European consortium collaborating in this project comprises most of the world's scientific leaders in this area, its results will have impact on the European biotech industry, since scientific success in the proposed project will be pursued further to develop agents that can be used in the clinical setting for modifying the system. This will involve small-to-medium sized European enterprises (biotech companies) that will collaborate on taking the findings of the project to further preclinical and clinical development. It is therefore of utmost importance to secure proprietary rights in every possible case.
The outcomes of the project will not only have impacts related to the identification of a possible new treatment target for osteoporosis, but the genetic analyses and the in vitro studies of the effect of genetic variations in bone cells have given significant insight into the pathogenesis and aetiology of osteoporosis. Further consolidating this knowledge about the genetic variations in the purinergic receptors and the association with osteoporosis may be used to identify individuals with a high propensity to develop osteoporosis. This means that identifying these patients at an early stage could lead to early treatment of the disease in such high risk patients. This would further impact the quality-of-life of the patients as well as the costs for fracture treatment in the health care system.
Other impacts evolving from the project are an increased awareness of osteoporosis in the public, as the dissemination measures taken by the consortium involves both press releases, contact with patient organisations, and a website addressing different aspects of osteoporosis and bone biology. Patients will also be aware of the many efforts taken to improve therapeutic approaches of the disease. Moreover, the level of awareness in politicians and decision makers of the epidemic size of the osteoporosis problem has and will also increase. This would be of major importance to influence the decision-making and allocation of health care resources to measures aimed at improving osteoporosis diagnosis and treatment. Thus a self-enforcing process has presumably been initiated by the project contributing to the wellness of European citizens and the society.
Steps needed to bring about the impact
The ATPBONE project was designed to take a major step forward in elucidating essential aspects of the P2 receptor signalling mechanisms involved in the regulation of bone formation and homeostasis, within the relatively limited time of the project. Thus, all aspects of the signalling system have been addressed. The project started with a basic characterisation of the signalling molecule (ATP) with respect to release mechanisms and stimuli, degradation and conversion. Next the expression pattern as well as the in vitro functions of the P2 receptors were addressed, to fully elucidate their role in osteoblast activity (matrix formation and mineralisation), as well as in osteoclast formation and resorptive activity in order to characterise bone homeostasis. Also the intracellular pathways activated by the receptor were investigated. To test the physiological significance and role of the signalling system, a number of genetically modified mouse models of osteoporosis were examined. Based on the findings from the laboratory and mouse studies, pre-clinical studies were performed demonstrating the usefulness of the system to modify bone formation and homeostasis in vivo by treatment with pharmacological agents active at the receptors.
As indicated above, the work plan of this project addressed all aspects necessary to fully elucidate the role of purinergic signalling in bone, as well as the potential of the purinergic signalling system in the treatment of osteoporosis in vivo. However, it was outside the scope and budget constraints to conduct clinical studies in humans as clinical trials require significantly more resources than available at the moment.
As evidenced by the number of different studies involved in the elucidation of signalling pathways, it was impossible to perform the current project in only one country. It was therefore of utmost importance to perform the project as a European collaborative project, not only to create the synergy between the participating scientific groups, but also to increase the awareness and impact in the whole EU instead of regionally or nationally.
In summary, the results from the ATPBONE project has the potential:
1. To contribute to an improved health and quality of life of the European citizens, osteoporosis patients especially, by delaying or preventing osteoporosis-related fractures.
2. To have a favourable impact on the economy of the community and healthcare systems, by affecting the competitiveness of the health-related industries or businesses especially small and medium-sized entreprises (SMEs) in the EU.
Dissemination and exploitation of project results
The results from the project are potentially of benefit to the public by providing new knowledge that may lead to new anabolic treatment strategies for osteoporosis, and therefore the dissemination of these results is of interest to the European citizens. The citizens of Europe have financed the project through taxes, and therefore have the right to be informed of the results and how the money is spent. The consortium has therefore taken measures to secure that the results from the project are widely disseminated to the public and to the scientific community. These measures include, but are not limited to the following:
- a website: http://www.atpbone.eu
- press releases describing important progress and findings in the project;
- contact with national osteoporosis patient organisations for articles in the membership magazines, and oral presentations at meetings in these societies.
The consortium has also taken measures to disseminate the results from the project to the scientific world, so that other scientists can use the acquired knowledge. This has taken place by:
- publication of the results in high rank international peer-reviewed scientific journals;
- oral and / or poster presentations at international scientific meetings and conventions
Thus the results from the project have been and will be disseminated to both the public, so that the European citizens will be informed about recent advances in osteoporosis treatment and aetiology as well as about the use of their tax money. Dissemination of the results to the scientific world will secure that the results can be used in the widest possible way to increase the knowledge about osteoporosis and bone biology, and to increase the chances that the findings will lead to improvement of treatment and handling of osteoporosis in the future.
Dissemination activities to scientific community:
During the project period, the ATPBONE consortium members have taken a number of measures to disseminate the knowledge emerging from the project. Naturally, there is a strong focus of disseminating the results from the scientific experiments to the academic world and scientific community in order to spread the highly interesting findings so that other can also build new experiments and knowledge on the data. Until now, 26 articles have been published already three years after the initiation of the project. Data analysis for most of the studies performed is still ongoing and it is estimated that an additional 50 peer reviewed scientific papers will be published within the next two years after the conclusion of the project period. Also, the scientists in the consortium have also been highly active in disseminating the results at national and international scientific meetings. A large number of abstracts have been submitted to these meeting, and many more are expected. Several of the abstracts have been selected for oral presentations and some of these have even been awarded prizes and awards. The focus of the ATPBONE project has generated significant interest within the scientific community. First, the consortium was asked to provide a number of review articles to a special issue of Frontiers in Bioscience, where also other leading non-European experts in the field of purinergic signalling in bone were invited to participate. Furthermore, based on the results of the ATPBONE project, the European Calcified Tissue Society has decided to have a special session on purinergic signalling in bone at the next annual international meeting in Stockholm in May 2012 with two of the ATPBONE members as keynote speakers. Thus, even with a lot of data still remaining to present from the project there has been a high interest from the scientific community.
Within the consortium we have taken a special step to increase the exchange of knowledge between groups but also to promote the ATPBONE project in the scientific community. Every six months, we have arranged an ATPBONE symposium. This symposium has moved around among the seven participating institutions, and has included presentations both by the principal investigators, young scientists from the participating centres as well as invited international capacities within the bone field. These symposia have received much interest from also outside the consortium and have been well attended. Furthermore, it is planned to continue these symposia also after the conclusion of the project, where annual meetings are planned for the exchange of ideas and for the continued collaboration among the partners. The ATPBONE symposia have also provided grounds for building a strong scientific network throughout the EU. Both senior and younger scientists have participated in the meeting, and important social activities have also been part of the meetings. This has increased the contact between both older and younger scientist with the added benefit of strengthening the network for the future of these scientists. This has been important and has also consolidated the future collaboration within the group and among all involved scientists.
Dissemination activities to target groups:
However, as it is important to promote and disseminate the findings also to other groups, the consortium has taken a number of measures to also disseminate the existence and findings from the project to other target groups, such as policy makers and the civil society. First, we have invested a great deal of time and human resources in promoting both the project as well as the knowledge of the European Commission (EC) and FP7 to other researchers, policy makers and other relevant persons. The coordinating center has engaged in a collaboration with the Danish contact point, Eurocenter, to teach at courses and information meetings for the scientific community and the industry to promote FP7. We have also been active in disseminating the knowledge of the project to the general public and to patients. During the course of the project, there have been several articles and interviews focusing on the project in patient magazines, magazines for employees of the health care system etc. In addition, we have one major article planned for publication in May in the magazine of the Danish Osteoporosis Organization (patient organisation), and another has already been published in the magazine of the Dutch Osteoporosis Organization. In addition, all consortium partners are focused on contacting the national osteoporosis organisations to have similar publications describing the ATPBONE project so that all relevant patients acquire knowledge about the project and the possible future implications.
Other measures have also been taken to spread the knowledge of the project. These have included oral presentations in patient organisations, participation in and presentation of the projects at science fairs (University of Ferrara) for adolescents, where the work package leader was awarded the best project presented.
Exploitation
The results from the project can potentially generate proprietary rights that need to be secured by patent applications. Throughout the duration of the project the consortium has constantly focused on whether there were patentable ideas and results that need to be secured. Until now, due to the relatively basic nature of the project, there have not been any patentable findings yet. However, the results obtained are highly interesting and further studies are planned to support potential patent applications and further exploitation of the foreground. As soon as it is relevant and the necessary amount of supporting data becomes available the participants in the consortium will take steps to determine whether a patent application is appropriate. The collaborators will also decide which participants have been involved in the patentable ideas / results.
List of websites:
For more information on the project and the consortium, please see the ATPBONE webpage at http://www.ATPBone.eu or contact scientific coordinator Niklas Rye Jørgensen (niklas@dadlnet.dk via e-mail).
