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Redundancy and complementarity of osteopontin and bone sialoprotein in skeletal biology – generation and experimental analysis of double knockout mice.

Description: Multiple factors, systemic and local, concur to the biomechanical competence of the bone mineralized matrix, which is a major determinant of bone strength. Matrix components play a key role, and in particular the proteins of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoproteins) family. Among these, bone sialoprotein (BSP) and osteopontin (OPN) are highly expressed by osteoblasts, hypertrophic chondrocytes and osteoclasts. Single knock-out (KO) of their respective genes have shown them to be key regulators of the process of bone formation, mineralization and turnover, with distinct, although partly overlapping sets of functions. However, given their structure similarities and coexpression in various cell types, partial functional substitution/compensation in a single KO model by the cognate protein is likely, and will impair proper understanding of the redundancy and complementarity of their roles in bone metabolism and mechanical resistance. Indeed, the study of SIBLING functions through the independent analysis of each gene has reached its limits, and the understanding of interplay between these factors now requires multiple targeting approaches. Our goal in this project is to analyse mice with a double extinction (DE) of OPN (Spp1 gene) and BSP (Ibsp gene), to describe their skeletal biology with multiple experimental approaches, comparing it with single extinctions of either gene, generated in the same genetic background. To generate DE mice, we shall use a BSP KO mouse line on a mixed, outbred 129sv/CD1 background, as well as mice produced by the EUCOMM consortium on the C57-Bl/6 genetic background, which display an insertion in the first intron of the Ibsp (BSP) gene that extinguishes its expression (BSPtme1). Using targeted mutagenesis with TALENs, we shall realise a KO of the Spp1 gene in BSP KO and BSPtme1 one-cell embryos, then generate through a genetic screening and breeding program, mice expressing neither BSP nor OPN (DE mice), as well as single OPN-/- mice on the same genetic background as the DE. The mouse lines will then be used to study in the same genetic background the impact of single and double mutation of OPN and BSP on major aspects of skeletal physiology and disease in which these two proteins have been shown to be involved, namely : (a) the bone turnover and (b) the bone microenvironment (vascularisation, haematopoiesis) and their response to challenges, (c) bone marrow ablation and (d) bone mechanical stimulation. (e) The bone mineralised matrix structure and composition as well as (f) its biomechanical resistance, will also be studied, and their response to intense mechanical strain (hypergravity) will be analysed. This work will be performed by two laboratories which have already collaborated in recent years to the elucidation of the BSP KO phenotype and collectively master the diversity of expertise necessary to this endeavour. Overall, this project will be a breakthrough in the elucidation of the respective roles of SIBLING proteins, which appear more and more as major regulators of bone and mineral metabolism, and are involved in an increasing number of other physiological mechanisms.

Funder: Agence Nationale de la Recherche

Amount: €295,000

Funding type: research

Funder scheme: Blanc 2013

Start date: 2014-01-01

Investigator: Luc MALAVAL

Institution: Laboratoire de biologie intégrative du tissu osseux

Topic: Bone and Dental Protein Studies

Subfield: Biochemistry

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Funded outputs count: 4