Final Report Summary - TALOS (Targeting LRP5 to Increase Bone Formation in Osteoporosis)
The aim of the TALOS project is to perform human, animal and basic studies to elucidate the role of LRP5 signaling and its antagonists on bone formation with final objective the identification and experimentally testing molecular targets for the eventual development of bone forming therapies for osteoporosis. This is a joint research effort of six academic and three industrial partners in the area of bone diseases. The main objectives of the project were the clinical, biochemical and molecular characterization of patients with craniotubular hyperostosis, rare bone disorders associated with excessive bone overgrowth, the identification of the mechanism of action of the bone formation inhibitor sclerostin and the synthesis of protein mimics that interfere with the slerostin-LRP5 interactions.
We fully characterised the clinical features of patients with sclerosteosis in South Africa and of patients with van Buchem disease in the Netherlands and defined the role of aging in the development of complications to the disease. By comparison to heterozygous disease carriers and controls we provided in vivo evidence of the importance of the absence or decreased synthesis of sclerostin on bone formation and bone quality in humans and we showed that inhibition of sclerostin can be titrated and elevate bone formation without causing any of the symptoms and complications of sclerosteosis. In addition, we proposed for the first time a medical treatment for patients with progressive, life-threatening increase in intracranial pressure resulting from the excessive growth of the bones of the skull. In a set of patients with craniotubular hyperostosis, DNA analysis of the SOST gene and of the first propeller of LRP5 identified new cases of sclerosteosis in patients of Brazilian origin, the first missense mutation of the SOST gene in a Turkish family and the first deletion of the LRP5 gene causing increased bone mass in a patient with the clinical picture of osteopetrosis. In addition, we screened these patients for mutations of other genes associated with the Wnt signaling pathway and we identified LRP4 as a sclerostin partner in facilitating the inhibitory action of LRP4 on bone. In cohorts of well characterised healthy women and their daughters and sons we showed that SOST gene polymorphisms influence bone remodeling, particularly after estrogen deprivation.
These results were in line with histomorphometric analysis of femoral neck biopsies from patients with osteoporosis, osteoarthritis and controls showing that osteonal sclerostin is a strong determinant of active bone formation by the osteoblasts and is markedly reduced during the inflammatory phase of fracture repair. In functional in vitro and in vivo studies of factors controlling LRP5 signaling we characterized the role of N-cadherin as a negative regulator of Wnt signaling and demonstrated that the matricellular protein periostin plays an important role in determining bone mass and microarchitecture in response to loading by both sclerostin-dependent and sclerostin-independent effects on osteoblast function. We determined the structure of sclerostin and we synthesised highly purified active full-length murine sclerostin as well as a large number of sclerostin proteins and mutant peptides which were used further to study the mechanism of action of sclerostin and to generate sclerostin-mimicking peptides and antibodies. We identified two regions in the sclerostin molecule required for inhibition of Wnt signaling, the loop region covering the loop tip and the finger 2 / cystine knot region, showing that binding of sclerostin to LRP5/6 alone is not sufficient for its inhibitory action on bone formation and underscoring the existence of a co-receptor for its action. In an analysis of putative co-receptors that mediate the inhibition of LRP5 signaling by sclerostin we identified LRP8 as a positive regulator of Wnt signaling and of Wnt-induced osteoblast differentiation which functions as a putative co-receptor of sclerostin. Finally, sclerostin mimicking peptides were synthesized in large quantities for in vivo studies and two of them were tested for their effects on bone mass and structure in animal model and in relation to loading. However, when given daily these peptides did not exert a clear anabolic effect on the skeleton.
Potential impact:
The European Commission (EC) strategy for life sciences and biotechnology has three main strategic aims: to meet societal needs, to increase competitiveness and to enhance European added value. The TALOS project can potentially impact on all these aims. Societal needs bone diseases are very common and are associated with significant morbidity, deterioration of quality of life and mortality. This is illustrated by the following examples. Osteoporosis is a chronic disabling disease of the skeleton that affects a large part of the ageing population in Europe. It is characterised by low bone mass and a deterioration in bone architecture leading to increased bone fragility and increased risk of fractures. Fractures are the main clinical consequence of the disease and their incidence increases with age. Approximately, 50 % of women who reach the age of 50 years are expected to suffer an osteoporotic fracture in the remaining part of their life. The annual incidence of fractures in the European Union (EU) is higher than 1.5 million and this is associated with considerable morbidity and mortality and escalation of health costs. Malignant disease account for about 20 % of all deaths in developed countries and certain cancers, such as breast cancer in women and prostate cancer in men, have a specific preference for the skeleton. Bone metastases are mainly associated with pain, fractures and neurological complications. Paget's disease is a skeletal disease that affects one or more bones, it progresses slowly and leads to changes in the shape and size of affected bones and to skeletal, joint and vascular complications. In parts of Europe, it is the second most common bone disorder affecting 2 to 5 % of people above the age of 50 years. With the increase in life expectancy of the population, the prevalence of bone diseases is expected to dramatically increase in the coming years. Disturbances in bone remodelling balance constitute the pathophysiological basis of these common skeletal disorders.
Understanding, therefore, the factors that regulate bone remodelling is essential for the management of patients with bone disease. Although considerable progress has been made in recent years in understanding the process of bone resorption, our knowledge of the regulation of bone formation remains inadequate. Consequently, the main stay of therapy of osteoporosis is agents that decrease bone resorption and maintain bone mass and architecture. However, these agents cannot build bone. For this, therapies that can stimulate bone formation are needed. A recent breakthrough has been the identification of the molecular defects in humans with rare skeletal disorders characterized by increased bone mass of good quality. Such diseases are the focus of the TALOS project.
The project succeeded in providing a better understanding of the function of components of the Wnt signaling pathway, a cell-signalling system in bone forming cells. This was achieved by the study of two rare bone disorders, sclerosteosis and Van Buchem disease, respectively, which are due to decreased secretion of the protein sclerostin, a negative regulator of bone formation. Most importantly, we found that in heterozygous carriers of the disease sclerostin synthesis is decreased and, compared to healthy subjects, this is associated with increased bone formation and bone mass. Remarkably these patients had none of the symptoms and complications of homozygous patients with sclerosteosis.
These findings strongly suggest that sclerostin synthesis can be titrated leading to increased bone formation without complications and open the way for clinical studies of patients with osteoporosis treated with inhibitors of sclerostin, some of which are currently under way. In addition, we provided for the first time a medical treatment for patients with these rare bone diseases and life-threatening complications resulting from excessive overgrowth of the skull bones. Finally, our studies elucidated the chemistry, molecular mechanism of action and regulation of secretion of sclerostin as well as its importance for bone mass and structure and identified additional targets for future therapeutic interventions. Thus, our results have an impact not only on elderly populations with increased bone fragility but also for patients with rare bone diseases and life-threatening complications.
This project is unique in bringing together groups with different expertises that are currently among the leaders in research in this research area internationally. Enhanced competition in the European life science and biotechnology sector is crucial for the European effort to lead biomedical research. The TALOS consortium provided a critical mass of complementary expertise that is necessary to address the objectives of the project and can ensure the leading position of Europe in this area. The innovative approaches of the project emerging from the close collaboration of academic institutions with three small and medium-sized enterprises (SMEs) can further enhance the European profile in biomedical research. As stated above, the proposed project addressed research efforts that were fragmented and uncoordinated internationally. The complexity of the topic required a high degree of networking between experts in the field. The TALOS project achieved that at the European level.
In addition, our consortium has access to the largest number of patients known with these rare bone disorders internationally. The TALOS project represents European added value:
a) because no single group could tackle the problem alone; and
b) because of the synergism in research efforts achieved.
Results of our studies have already been published in international journals and a number of manuscripts have been either submitted for consideration of publication or are in preparation. In addition, the scientific activities of the members of the consortium have been presented in international and national congresses (41 presentations in total) or have been communicated to public media. A web site was developed (see http://www.talosproject.nl(opens in new window) online) which contains a public and a protected domain for the use of the members of the consortium.
During the project period members of the consortium met biannually for two days in Leiden. Importantly, these meetings were not only attended by the members of the General Assembly but also by younger investigators working with the various beneficiaries, providing additional educational value to the discussions on the project. Detailed reports of all meetings were prepared and slides of the presentations were loaded on the web site for use by all participants. In addition, there has been intensive, project-based contact between individual members of the consortium. Characteristic of the quality of the data generated is the Young Investigator Award awarded to Dr A van Lierop at the congress of the ECTS/IBMS 2011 for his work on circulating sclerostin in humans, the prize for best oral presentation at the annual meeting of the Dutch Society for Calcium and Bone Metabolism and the President's prize for best poster at the ASBMR Congress 2011. In addition, members of the consortium involved in teaching, discussed several aspects of bone diseases and in particular of bone sclerosing dysplasias with undergraduate and graduate students. The consortium has also been active in the public domain.
A meeting was held in Urk, the Netherlands, where nearly all known patients with Van Buchem disease live, which was attended by patients and their families as well as local general practitioners and an ENT surgeon specialized in operations of such patients. During this meeting we had the opportunity to discuss in depth various aspects of the disease and to present our research program which was very positively received. Patients and family members further agreed to participate in this research effort. Following this original meeting in Urk, the Netherlands, with patients with Van Buchem Disease, we had further discussions with this group which led to the successful recruitment of a considerable number of individuals for our studies.
In addition, a leaflet with information about the disease was prepared and was distributed to patients and family members as well as GPs of Urk. In a half page interview by one of the major daily newspapers of Greece (Kathimerini, 23 April 09) the coordinator discussed the various aspects of the project emphasizing its multidisciplinary European structure made possible by the EC funding. This was followed by a presentation of the project in the evening news of a national Greek TV channel (Skai TV) and additional interviews by another Greek newspaper (Ethnos) and by the magazine of LUMC (Cicero; circulation about 10 000).
Project website: http://www.talosproject.nl(opens in new window)
We fully characterised the clinical features of patients with sclerosteosis in South Africa and of patients with van Buchem disease in the Netherlands and defined the role of aging in the development of complications to the disease. By comparison to heterozygous disease carriers and controls we provided in vivo evidence of the importance of the absence or decreased synthesis of sclerostin on bone formation and bone quality in humans and we showed that inhibition of sclerostin can be titrated and elevate bone formation without causing any of the symptoms and complications of sclerosteosis. In addition, we proposed for the first time a medical treatment for patients with progressive, life-threatening increase in intracranial pressure resulting from the excessive growth of the bones of the skull. In a set of patients with craniotubular hyperostosis, DNA analysis of the SOST gene and of the first propeller of LRP5 identified new cases of sclerosteosis in patients of Brazilian origin, the first missense mutation of the SOST gene in a Turkish family and the first deletion of the LRP5 gene causing increased bone mass in a patient with the clinical picture of osteopetrosis. In addition, we screened these patients for mutations of other genes associated with the Wnt signaling pathway and we identified LRP4 as a sclerostin partner in facilitating the inhibitory action of LRP4 on bone. In cohorts of well characterised healthy women and their daughters and sons we showed that SOST gene polymorphisms influence bone remodeling, particularly after estrogen deprivation.
These results were in line with histomorphometric analysis of femoral neck biopsies from patients with osteoporosis, osteoarthritis and controls showing that osteonal sclerostin is a strong determinant of active bone formation by the osteoblasts and is markedly reduced during the inflammatory phase of fracture repair. In functional in vitro and in vivo studies of factors controlling LRP5 signaling we characterized the role of N-cadherin as a negative regulator of Wnt signaling and demonstrated that the matricellular protein periostin plays an important role in determining bone mass and microarchitecture in response to loading by both sclerostin-dependent and sclerostin-independent effects on osteoblast function. We determined the structure of sclerostin and we synthesised highly purified active full-length murine sclerostin as well as a large number of sclerostin proteins and mutant peptides which were used further to study the mechanism of action of sclerostin and to generate sclerostin-mimicking peptides and antibodies. We identified two regions in the sclerostin molecule required for inhibition of Wnt signaling, the loop region covering the loop tip and the finger 2 / cystine knot region, showing that binding of sclerostin to LRP5/6 alone is not sufficient for its inhibitory action on bone formation and underscoring the existence of a co-receptor for its action. In an analysis of putative co-receptors that mediate the inhibition of LRP5 signaling by sclerostin we identified LRP8 as a positive regulator of Wnt signaling and of Wnt-induced osteoblast differentiation which functions as a putative co-receptor of sclerostin. Finally, sclerostin mimicking peptides were synthesized in large quantities for in vivo studies and two of them were tested for their effects on bone mass and structure in animal model and in relation to loading. However, when given daily these peptides did not exert a clear anabolic effect on the skeleton.
Potential impact:
The European Commission (EC) strategy for life sciences and biotechnology has three main strategic aims: to meet societal needs, to increase competitiveness and to enhance European added value. The TALOS project can potentially impact on all these aims. Societal needs bone diseases are very common and are associated with significant morbidity, deterioration of quality of life and mortality. This is illustrated by the following examples. Osteoporosis is a chronic disabling disease of the skeleton that affects a large part of the ageing population in Europe. It is characterised by low bone mass and a deterioration in bone architecture leading to increased bone fragility and increased risk of fractures. Fractures are the main clinical consequence of the disease and their incidence increases with age. Approximately, 50 % of women who reach the age of 50 years are expected to suffer an osteoporotic fracture in the remaining part of their life. The annual incidence of fractures in the European Union (EU) is higher than 1.5 million and this is associated with considerable morbidity and mortality and escalation of health costs. Malignant disease account for about 20 % of all deaths in developed countries and certain cancers, such as breast cancer in women and prostate cancer in men, have a specific preference for the skeleton. Bone metastases are mainly associated with pain, fractures and neurological complications. Paget's disease is a skeletal disease that affects one or more bones, it progresses slowly and leads to changes in the shape and size of affected bones and to skeletal, joint and vascular complications. In parts of Europe, it is the second most common bone disorder affecting 2 to 5 % of people above the age of 50 years. With the increase in life expectancy of the population, the prevalence of bone diseases is expected to dramatically increase in the coming years. Disturbances in bone remodelling balance constitute the pathophysiological basis of these common skeletal disorders.
Understanding, therefore, the factors that regulate bone remodelling is essential for the management of patients with bone disease. Although considerable progress has been made in recent years in understanding the process of bone resorption, our knowledge of the regulation of bone formation remains inadequate. Consequently, the main stay of therapy of osteoporosis is agents that decrease bone resorption and maintain bone mass and architecture. However, these agents cannot build bone. For this, therapies that can stimulate bone formation are needed. A recent breakthrough has been the identification of the molecular defects in humans with rare skeletal disorders characterized by increased bone mass of good quality. Such diseases are the focus of the TALOS project.
The project succeeded in providing a better understanding of the function of components of the Wnt signaling pathway, a cell-signalling system in bone forming cells. This was achieved by the study of two rare bone disorders, sclerosteosis and Van Buchem disease, respectively, which are due to decreased secretion of the protein sclerostin, a negative regulator of bone formation. Most importantly, we found that in heterozygous carriers of the disease sclerostin synthesis is decreased and, compared to healthy subjects, this is associated with increased bone formation and bone mass. Remarkably these patients had none of the symptoms and complications of homozygous patients with sclerosteosis.
These findings strongly suggest that sclerostin synthesis can be titrated leading to increased bone formation without complications and open the way for clinical studies of patients with osteoporosis treated with inhibitors of sclerostin, some of which are currently under way. In addition, we provided for the first time a medical treatment for patients with these rare bone diseases and life-threatening complications resulting from excessive overgrowth of the skull bones. Finally, our studies elucidated the chemistry, molecular mechanism of action and regulation of secretion of sclerostin as well as its importance for bone mass and structure and identified additional targets for future therapeutic interventions. Thus, our results have an impact not only on elderly populations with increased bone fragility but also for patients with rare bone diseases and life-threatening complications.
This project is unique in bringing together groups with different expertises that are currently among the leaders in research in this research area internationally. Enhanced competition in the European life science and biotechnology sector is crucial for the European effort to lead biomedical research. The TALOS consortium provided a critical mass of complementary expertise that is necessary to address the objectives of the project and can ensure the leading position of Europe in this area. The innovative approaches of the project emerging from the close collaboration of academic institutions with three small and medium-sized enterprises (SMEs) can further enhance the European profile in biomedical research. As stated above, the proposed project addressed research efforts that were fragmented and uncoordinated internationally. The complexity of the topic required a high degree of networking between experts in the field. The TALOS project achieved that at the European level.
In addition, our consortium has access to the largest number of patients known with these rare bone disorders internationally. The TALOS project represents European added value:
a) because no single group could tackle the problem alone; and
b) because of the synergism in research efforts achieved.
Results of our studies have already been published in international journals and a number of manuscripts have been either submitted for consideration of publication or are in preparation. In addition, the scientific activities of the members of the consortium have been presented in international and national congresses (41 presentations in total) or have been communicated to public media. A web site was developed (see http://www.talosproject.nl(opens in new window) online) which contains a public and a protected domain for the use of the members of the consortium.
During the project period members of the consortium met biannually for two days in Leiden. Importantly, these meetings were not only attended by the members of the General Assembly but also by younger investigators working with the various beneficiaries, providing additional educational value to the discussions on the project. Detailed reports of all meetings were prepared and slides of the presentations were loaded on the web site for use by all participants. In addition, there has been intensive, project-based contact between individual members of the consortium. Characteristic of the quality of the data generated is the Young Investigator Award awarded to Dr A van Lierop at the congress of the ECTS/IBMS 2011 for his work on circulating sclerostin in humans, the prize for best oral presentation at the annual meeting of the Dutch Society for Calcium and Bone Metabolism and the President's prize for best poster at the ASBMR Congress 2011. In addition, members of the consortium involved in teaching, discussed several aspects of bone diseases and in particular of bone sclerosing dysplasias with undergraduate and graduate students. The consortium has also been active in the public domain.
A meeting was held in Urk, the Netherlands, where nearly all known patients with Van Buchem disease live, which was attended by patients and their families as well as local general practitioners and an ENT surgeon specialized in operations of such patients. During this meeting we had the opportunity to discuss in depth various aspects of the disease and to present our research program which was very positively received. Patients and family members further agreed to participate in this research effort. Following this original meeting in Urk, the Netherlands, with patients with Van Buchem Disease, we had further discussions with this group which led to the successful recruitment of a considerable number of individuals for our studies.
In addition, a leaflet with information about the disease was prepared and was distributed to patients and family members as well as GPs of Urk. In a half page interview by one of the major daily newspapers of Greece (Kathimerini, 23 April 09) the coordinator discussed the various aspects of the project emphasizing its multidisciplinary European structure made possible by the EC funding. This was followed by a presentation of the project in the evening news of a national Greek TV channel (Skai TV) and additional interviews by another Greek newspaper (Ethnos) and by the magazine of LUMC (Cicero; circulation about 10 000).
Project website: http://www.talosproject.nl(opens in new window)
