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

Generation and experimental analysis of the double gene silencing of osteopontin (OPN) and bone sialoprotein (BSP) in mice.

The experimental program aims at comparing in mice with single and double knockout of BSP and OPN, (1) the basic skeletal phenotype, (2) its response to an experimental bone reapir challenge (marrow ablation) and (3) its response to two protocols of mechanical stimulation (vibrations, hypergravity).

Redundancy and complementarity of OPN and BSP in skeletal biology

Proteins of the SIBLING («Small Integrin Binding Ligand N-linked Glycoproteins«) family are bone extracellular matrix components that play a major role in skeletal biology and in particular its biomechanical competence. Among them, BSP and OPN are highly expressed by bone cells. The gene extinction of BSP or OPN induce distinct bone phenotypes, suggesting separate functions. However, our previous work showed at least partial functional substitution between these two proteins (Bouleftour et al., 2015), which complicates the understanding of their respective roles in bone physiology. The generation of double-KO mice for these proteins provides a valuable tool to investigate these issues.

1 / mice with dual extinction (DE) off OPN and BSP genes (SPP1 -/- IBSP -/-) were generated by injection of TALE nucleases (TALEN) in mouse ova after in vitro fecundation.
2 / The exploration of the skeletal phenotype of mutant mice uses microtomography, vascular imaging, histomorphometry, cytometry, immunohistochemsitry and QRT-PCR.
3 / The ability of mouse lines to repair their bones will be analyzed in a marrow ablation model.
4 / The effects of mechanical stimulation will be analyzed with vibrating plates and a centrifugation device generating a hypergravity.
4 / The effect of the mutations on the composition of the mineralized matrix and its biomechanical properties, and the impact of hypergravity on these parameters, will be analyzed by resistance testing (4 points bending), FTIRI, quantitative microradiograph and biochemistry .

Mice DE reach adulthood and are fertile, showing that dual KO of OPN and BSP is not lethal. They have a normal morphology, but lower size and weight than wild mice, as is the case for the BSP-/- (. Malaval et al, 2008) but not for OPN -/- mice.
The microtomographic analysis of showed that DE bones have a lower trabecular volume (BV/TV) than wild type mice, while that of BSP-/- is higher (. Malaval et al, 2008) . The mineral density of the cortical bone of mice DE is lower than that of wild type, like the BSP -/-.

The lower mineral density of DE bones is counter-intuitive, because OPN is a factor inhibiting mineralization. It suggests the existence of complex interactions between BSP and OPN in the development of the bone mineral, and justifies the interest of the model for their study.

Overall, this work in progress will lead to a better understanding of the respective roles in skeletal biology of OPN and BSP, two members of a family (the SIBLING) whose evolutionary appearance coincides with the development of endochondral bone.

The generation of DE mice was the subject of an oral communicationat the 16ièmes JFBTM, Limoges, 14-16 May 2014 and a poster presentation at the «12th Transgenic Technology Meeting« in Edinburgh, 6-18 October 2014 .

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.

Project coordinator

Monsieur Luc MALAVAL (Laboratoire de biologie intégrative du tissu osseux)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INSERM U1059/LBTO Laboratoire de biologie intégrative du tissu osseux
INSERM U1033 Physiopathologie, Diagnostic et traitement des maladies osseuses

Help of the ANR 294,999 euros
Beginning and duration of the scientific project: December 2013 - 42 Months

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