EphB/ephrin-B interactions assist in MSC niche maintenance and contribute to bone remodelling following injury

Abstract

Category 6. Bone formation, cartilage and bone matrix 401 Keratin 18 is over-expressed in osteoblasts derived from Pagetic lesions but is not involved in the formation of disrupted matrix D. Naot, B.G. Matthews, R.M. Locklin, Z. Xia, P.A. Hulley, J. Cornish Department of Medicine, University of Auckland, Auckland, New Zealand Nuffield Department of Orthopaedic Surgery, Botnar Research Centre, Oxford, United Kingdom Paget's disease is a common focal bone disorder. Pagetic lesions, which result from over-activity of osteoclasts and osteoblasts, appear lytic at early stages and later turn sclerotic, with areas of irregular, disorganised bone matrix. Pagetic osteoclasts are grossly abnormal, and have been the focus of most of the research on the cellular mechanisms of Paget's. Given the tight coupling between osteoclasts and osteoblasts, we characterised changes in the pagetic osteoblast that could contribute to the development of the disease. We compared gene expression in osteoblasts and bone marrow cells from pagetic and non-pagetic patients. Microarray analysis identified a number of differentially regulated genes, and the intermediate filament protein keratin 18 (KRT18) was one of the most highly upregulated genes in pagetic osteoblasts. Real-time RT-PCR comparing 28 pagetic samples to 49 controls confirmed that KRT18 expression is more than three times higher in pagetic cells. In the present study we investigated the effects of KRT18 over-expression on osteoblasts and mesenchymal cells. Primary human osteoblasts were transduced with a KRT18 adenoviral vector and compared to cells transduced with a control vector. Real-time RT-PCR showed that KRT18 over-expressing cells have increased levels of interleukin 1β, Dkk1, and the chemokine MCP1, genes that had previously been identified as up-regulated in pagetic osteoblasts. In order to study the possible effects of KRT18 over-expression on cell morphology and extracellular matrix formation, we cultured virally transduced human primary mesenchymal cells in 3-dimensional collagen scaffolds. The scaffolds provide an extracellular structure which resembles the bone environment and is therefore better suited for the study of cell morphology and matrix formation and mineralisation. Cells were analysed using confocal imaging. Cells were also stained for alkaline phosphatase activity and calcium deposition was measured by alizarin red staining. Cultures of KRT18 over-expressing cells were not significantly different from the controls. In conclusion, these results suggest that KRT18 plays a role in osteoblast biology, and overexpression of this gene can reproduce some of the features of pagetic osteoblasts, however, this does not appear to include the disrupted matrix formation. doi:10.1016/j.bone.2009.01.454 402 EphB/ephrin-B interactions assist in MSC niche maintenance and contribute to bone remodelling following injury A. Arthur, C. Stylianou, S.A. Koblar, K. Matsuo, S. Gronthos Bone and Cancer Laboratories, Division of Haematology, Institute of Medical and Veterinary Science/Hanson Institute, Adelaide, SA, Australia The Institute of Molecular and Cell Biology, Singapore's Agency for Science, Technology and Research, Singapore, Singapore School of Molecular and Biomedical Science, School of Medicine, University of Adelaide, Adelaide, SA, Australia Department of Microbiology and Immunology, Graduate School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan The bone marrow contains mesenchymal stromal/stem cells (MSCs) that reside within a perivascular niche. This precursor population is essential for regulating skeletal tissue homeostasis, including bone formation and repair. Bone remodelling is mediated by bone forming osteoblasts derived from MSC, and by bone resorbing osteoclasts that originate from the haematopoietic lineage. However, the molecular signals that maintain multi-potential MSC populations within the stem cell niche and the mechanisms that drive their mobilization towards the bone surfaces to facilitate bone formation are not well defined. The Eph/ephrin family of receptor tyrosine kinases have been implicated in the maintenance of stem cell niches including neural, intestinal and dental tissue, and more recently in the regulation of bone homeostasis. We have characterized the gene and protein expression of EphB/ephrin-B molecules on human culture expanded MSC populations and found that the EphB1, B2 and B4 receptors and ephrin-B1/B2 ligand were the highest expressed B class Eph/ephrin molecules. Functional studies using Fc fusion proteins bound to the extracellular portion of EphB2 or ephrin-B1 showed that reverse ephrin-B signalling inhibited human MSC attachment and spreading, while forward EphB signalling promoted MSC migration. Furthermore, EphB expressing MSC were found to restrict their capacity for selfrenewal while activated ephrin-B expressing MSC promoted osteogenic differentiation. A murine femoral fracture model was used to elucidate the contribution of EphB/ephrin-B molecules to skeletal tissue repair and bone remodelling. Gene expression for 8756–3282/$ – see front matter Bone 44 (2009) S142–S161