Sub theme 1.2
Regulation of calcium and bone metabolism

Goals of research: general outline
Scientific achievements
Future plans: special goals and approach
Running projects
Associated staff

Workgroup leaders   Department
dr.  J.P.T.M.  van  Leeuwen   Internal Medicine
PhD  E.L.  Lubberts   Rheumatology

Goals of research: general outline

The focus of this research program is on skeletal and calcium homeostasis and development and progression of related disorders with particular emphasis on aging. Increasing life span is accompanied by an increase in age-related diseases. In particular, osteoporosis (OP) and osteoarthritis (OA) affect the locomotor system and greatly limit mobility at advanced ages. Both OP and OA are important common age-related diseases with a large impact on quality of life as well as health care budget.

Development of pharmaceutical and life style interventions as prevention or as treatment is important and requires an optimal knowledge in aetiology and mechanisms of development of disease. Genetic and genomic studies can help to identify the genes and biological pathways involved, and thereby improve biological understanding of the disease process. Such studies can identify biological markers for (early) diagnostics and also allow for the development of novel, potentially therapeutic interventions.

Current therapies for OP are directed to inhibit bone loss and thereby its progression. However, because bone loss has already occurred when the consequences of OP have become overt this is not an optimal treatment. Thus, there is a major need for anabolic therapies, which stimulate bone formation and thereby improve bone health. Similarly, for OA no (optimal) therapies are available and the existing treatments are focussed on pain reduction and/or joint replacement. While OP and OA share some common biological and disease pathways and share target tissues, both are studied in this program. Traditionally this has been focussed on OP while OA research is more recent.

The department of Internal Medicine has collaborating combinations of molecular, genetic, cellular, animal, epidemiological and clinical studies on calcium and bone metabolism. It is our vision that not these individual approaches but that the combination of these approaches and integration of data sets is the key to success. Bio-informatic and systems biological analyses of gene, protein and enzyme activity profiles to characterise bone cell differentiation, -formation and -degradation using human bone cell models. This includes development of medium/high throughput screenings procedures to identify therapeutic targets and to evaluate novel therapeutic compounds. Calcium homeostasis and skeletal metabolism is also studied in relation to ageing by analyses of experimental animal models, e.g. premature ageing mice.

Scientific achievements

Molecular Cell Biological Research

Firstly, osteoblasts, the bone forming cells, play a pivotal role in bone metabolism by forming bone and controlling bone resorption by osteoclasts. In the clinic the therapies for osteoporosis are directed to inhibit bone resorption (i.e. inhibition of further bone loss). No potent bone anabolic therapies are available. We focussed on identifying bone anabolic therapeutic targets as well as novel diagnostic markers (paralleling the genetic approaches). For this we characterized a human osteoblast-based bone formation model as well as set up human mesenchymal stem cell studies (osteoblasts are derived from mesenchymal stem cells). We performed extensive gene expression profiling and protein profiling of these cell models against the backdrop of the osteoblast differentiation and bone formation. Within a EU program and a NWO funded TOP grant we focus to develop systems biological approaches to explain osteoblast differentiation and bone formation.

The expression profiling studies and perturbation studies with growth factors lead to a first identification of follistatin as a bone anabolic target. This finding and the identified changes in extracellular matrix gene expression in relation to mineralization led to patent applications and the founding of a spin off company: Therosteon. Currently there is a short list of drug/diagnostic target candidates that is further functionally screened and approaches are developed to merge the expression profiling data with the genome wide association data (see part B.)

Secondly, calcium homeostasis in relation to aging is studied at population level in the Rotterdam study and at animal level. Together with the Department of Physiology (Prof. Bindels) of the university of Nijmegen we were able to demonstrate a crucial role for the calcium channel TRPV5 in maintaining calcium homeostasis in which bone plays a prominent role. More recently we demonstrated that TPRV5 is also expressed in osteoclast and is involved in bone resorption. Currently studies (at cellular and transgenic animal level) are ongoing to identify the role of TPRV6 (like TRPV5 calcium specific) and TRPV4 (a non-calcium specific channel). Both are expressed in osteoblasts as well as osteoblasts. These studies will contribute to the knowledge of the role of these channels in bone metabolism and may provide clues for intervention.

Thirdly, together with the Genetics Department (Prof. Hoeijmakers and Prof. van der Horst) the skeletal phenotype and mesenchymal stem cell behaviour with aging as been investigated in DNA repair impaired transgenic animals (TTD mice). These animals show a normal skeletal development up to 39 weeks of age but after that demonstrate and strongly accelerated skeletal aging, which is preceded by a drop in mesenchymal stem cells. These studies demonstrated the significance of proper DNA repair for healthy aging. These data have been a strong support for DNage (biotech spin off by Prof. Hoeijmakers). This has led together with DNage to an European grant together with a Spanish company to develop a zebra fish based screening system of therapeutic and diagnostic candidates.

Future plans: special goals and approach

Current molecular cell biological work and aging studies will be taken forward. In addition gene and protein profiles will be combined by intersection analyses in relation to the phenotypic expression (i.e. bone formation). This way identified genes will be combined with GWAS data an functionally analysed for their impact on bone formation and used for potential drug development.  Focus of research will also be to apply systems biology approaches to model the development of stem cells with the aim to be able to better understand and control stem cell renewal, lineage commitment and differentiation.

In collaboration with Department of Hematology (Prof. J. Cornelissen) a research line will be developed to study the osteoblast stem cell niche. The aim is a.o. to be improve cord blood usage for bone marrow transplantation.

Together with the department of Rheumatology studies will be performed on vitamin D control of immune cell function and the interaction between immune cells, osteoclasts and osteoblasts.

Recent development is the setup of protein profiling as well as serum miRNA profiling of women with or without osteoporosis (low bone mass and fractures). The analyses are currently ongoing has been set up and these analyses are on going. The focus is identifying osteoporosis specific markers, which will be linked to the genetic studies. In addition, osteoclasts have been cultured from peripheral blood of these women and are used for functional comparison.

Most recent publications

1.      M Eijken, S Swagemakers, M Koedam, C Steenbergen, P Derkx, AG Uitterlinden, PJ van der Spek, JA Visser, FH de Jong, HA Pols, JPTM van Leeuwen. (2007) The activin A-follistatin system: potent regulator of human extracellular matrix mineralization. FASEB J. 2007 21(11):2949-60. IF:  6.79

2.      M van Driel, M Koedam, CJ Buurman, M Hewison, H Chiba, AG Uitterlinden, HAP Pols, JPTM van Leeuwen (2006) Evidence for auto/paracrine actions of vitamin D in bone: 1α-hydroxylase expression and activity in human bone cells. FASEB J Express, 20(13):2417-2419. IF: 6.79

3.      M.Eijken, M. Hewison, M.S. Cooper, FH de Jong, H Chiba, PM Stewart, AG Uitterlinden, HAP Pols, JPTM van Leeuwen. (2005) 11β-Hydroxysteroid dehydrogenase expression and glucocorticoid synthesis are directed by a molecular switch during osteoblast differentiation. Mol Endocrinology 19(3):621-631. IF 5.34

4.      BC van der Eerden, JG Hoenderop, TJ de Vries, T Schoenmaker, CJ Buurman, AG Uitterlinden, HAP Pols, RJ Bindels, JPTM van Leeuwen. (2005) The epithelial Ca2+ channel TRPV5 is essential for proper osteoclastic bone resorption. Proc Natl Acad Sci U S A.; 102(48):17507-17512. IF: 9.6

5.      JG Hoenderop, JPTM van Leeuwen, BC van der Eerden, FF Kersten, van der Kemp AW, Merillat AM, Waarsing JH, Rossier BC, Vallon V, Hummler E, Bindels RJ (2003) Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5. J Clin Invest 112(12):1906-1914. IF :16.92