Purpose: Matrix gla protein (Mgp) is a structural protein of the extracellular matrix, which belongs to the family of extracellular mineral-binding proteins called Gla proteins. Mgp is mainly synthesized and expressed by chondrocytes, vascular smooth muscle cells, endothelial cells and fibroblasts. If Mgp has been shown to play a critical role in preventing pathological mineralization of the vascular wall, its role in skeletal development has hardly been studied. In this context, the purpose of our research was to investigate the role of Mgp in skeletal growth and endochondral ossification.
Methods: Mgp wild-type and Mgp knock-out mice were obtained from the intercross of heterozygous parent mice. Mice from the same litter were analyzed and compared histomorphometrically by whole-mount Alizarin Red/Alcian Blue skeletal staining and histologically by specific stainings to measure growth plate zone length, and observe chondrocyte organization and mineralization and by in situ hybridization to detect specific markers expressed in the different zones of the growth plate. In parallel, primary immature chondrocyte cultures from Mgp wild-type and Mgp knock-out pups were analyzed for expression profiling of chondrocyte proliferation, differentiation and mineralization by quantitative PCR (qPCR).
Results: Whole-mount Alizarin Red/Alcian Blue skeletal preparations demonstrated that Mgp knock-out mice exhibit smaller skeleton size than Mgp wild-type mice at P21. Measurement of the skeleton elements, including femur, tibia, head, spine, sternum, showed and confirmed that dwarfism of Mgp knock-out mice is substantially noticeable at age P21. Alcian Blue and Safranin O stainings of the growth plate from embryonic stage (E14.5) to early postnatal stages before weaning (P21), demonstrated that a significantly shortened and disturbed hypertrophic zone in Mgp knock-out when compared to that of Mgp wild-type mice can already be observed around P6. In situ hybridization experiments further confirmed the decreased length of the hypertrophic zone (Col 10 positive) at P6 in Mgp knock-out mice. Expression profiling of primary immature chondrocytes demonstrated that the absence of Mgp in these cells significantly modulates the expression of proliferative chondrocyte markers (Col 2) and relative transcription factors (Sox9, Hif-1α, Hif-2α), hypertrophic markers (Col 10, OPN) and relative signaling molecules (BMP2, Ihh). Interestingly, the expression of genes implicated in the Pi/PPi balance (Ank, Enpp3, PC1, Pit-1, CD73) was also modified in Mgp-deficient chondrocytes.
Conclusions: Our study suggests that Mgp plays critical roles in regulating chondrocyte proliferation and the timing of maturation and mineralization during postnatal endochondral ossification.