Abstract
Deformities of cranial sutures such as craniosynostosis and enlarged parietal foramina greatly impact human development and quality of life. Here we have examined the role of the extracellular matrix protein ameloblastin (Ambn), a recent addition to the family of non-collagenous extracellular bone matrix proteins, in craniofacial bone development and suture formation. Using RT-PCR, western blot and immunohistochemistry, Ambn was localized in mouse calvarial bone and adjacent condensed mesenchyme. Five-fold Ambn overexpression in a K14-driven transgenic mouse model resulted in delayed posterior frontal suture fusion and incomplete suture closure. Moreover, Ambn overexpressor skulls weighed 13.2% less, their interfrontal bones were 35.3% thinner, and the width between frontal bones plus interfrontal suture was 14.3% wider. Ambn overexpressing mice also featured reduced cell proliferation in suture blastemas and in mesenchymal cells from posterior frontal sutures. There was a more than 2-fold reduction of Msx2 in Ambn overexpressing calvariae and suture mesenchymal cells, and this effect was inversely proportionate to the level of Ambn overexpression in different cell lines. The reduction of Msx2 expression as a result of Ambn overexpression was further enhanced in the presence of the MEK/ERK pathway inhibitor O126. Finally, Ambn overexpression significantly reduced Msx2 down-stream target gene expression levels, including osteogenic transcription factors Runx2 and Osx, the bone matrix proteins Ibsp, ColI, Ocn and Opn, and the cell cycle-related gene CcnD1. Together, these data suggest that Ambn plays a crucial role in the regulation of cranial bone growth and suture closure via Msx 2 suppression and proliferation inhibition.
Highlights
During skull development, calvarial bones expand from initial ossification centers toward the suture interface between adjacent bones [1]
This gradual intramembranous ossification is accomplished by the differentiation of ectomesenchymal cells into calvarial osteoblasts [2]
Membranous bone ossification is regulated by a number of growth and transcription factors as well as extracellular matrix proteins, including fibroblast growth factor receptors FGFR1 and FGFR2 [3], transcription factors CBFA1/RUNX2 [4,5,6], MSX2 [7], TWIST [8],[9], and matrix proteins osteopontin and tenascin [10],[11]
Summary
Calvarial bones expand from initial ossification centers toward the suture interface between adjacent bones [1]. Membranous bone ossification is regulated by a number of growth and transcription factors as well as extracellular matrix proteins, including fibroblast growth factor receptors FGFR1 and FGFR2 [3], transcription factors CBFA1/RUNX2 [4,5,6], MSX2 [7], TWIST [8],[9], and matrix proteins osteopontin and tenascin [10],[11] Abnormal changes in these factors affect bone thickness and mineralization, and the expansion of bone along the ossification center/suture axis, resulting in either synostosis or open foramina. Complications from surgical treatment can lead to infection, re-ossification, and death from hemorrhage [15]
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