Abstract
Bone sialoprotein (BSP) belongs to the "small integrin-binding ligand N-linked glycoprotein" (SIBLING) family, whose members interact with bone cells and bone mineral. BSP is strongly expressed in bone and we previously showed that BSP knockout (BSP-/-) mice have a higher bone mass than wild type (BSP+/+) littermates, with lower bone remodelling. Because baseline bone formation activity is constitutively lower in BSP-/- mice, we studied the impact of the absence of BSP on in vitro osteogenesis in mouse calvaria cell (MCC) cultures. MCC BSP-/- cultures exhibit fewer fibroblast (CFU-F), preosteoblast (CFU-ALP) and osteoblast colonies (bone nodules) than wild type, indicative of a lower number of osteoprogenitors. No mineralized colonies were observed in BSP-/- cultures, along with little/no expression of either osteogenic markers or SIBLING proteins MEPE or DMP1. Osteopontin (OPN) is the only SIBLING expressed in standard density BSP-/- culture, at higher levels than in wild type in early culture times. At higher plating density, the effects of the absence of BSP were partly rescued, with resumed expression of osteoblast markers and cognate SIBLING proteins, and mineralization of the mutant cultures. OPN expression and amount are further increased in high density BSP-/- cultures, while PHEX and CatB expression are differentiatlly regulated in a manner that may favor mineralization. Altogether, we found that BSP regulates mouse calvaria osteoblast cell clonogenicity, differentiation and activity in vitro in a cell density dependent manner, consistent with the effective skeletogenesis but the low levels of bone formation observed in vivo. The BSP knockout bone microenvironment may alter the proliferation/cell fate of early osteoprogenitors.
Highlights
The small integrin-binding ligand N-linked glycoprotein" (SIBLING) (Small Integrin Binding N-Linked Glycoproteins, [1]) are a family of extracellular matrix (ECM) targeted factors highly expressed in bone and dentin and which comprise Bone sialoprotein (BSP) (Bone Sialoprotein), OPN, matrix extracellular phosphoglycoprotein (MEPE) (Matrix Extracellular PhosphoglycoprotEin), DMP-1 (Dentin Matrix Protein-1) and DSPP (Dentin SialoPhosphoProtein)
CFU-F and CFU-alkaline phosphatase (ALP) numbers were assessed in BSP+/+ and-/- mouse calvaria cell (MCC) cultures plated at low density
BSP has long been considered a marker of late osteoblastic differentiation, the actual role(s) of the protein in bone biology remain poorly known
Summary
The SIBLING (Small Integrin Binding N-Linked Glycoproteins, [1]) are a family of extracellular matrix (ECM) targeted factors highly expressed in bone and dentin and which comprise BSP (Bone Sialoprotein), OPN (osteopontin), MEPE (Matrix Extracellular PhosphoglycoprotEin), DMP-1 (Dentin Matrix Protein-1) and DSPP (Dentin SialoPhosphoProtein). The SIBLING genes are aligned together in human chromosome 4 and mouse chromosome 5, in a region called the “bone gene cluster” [2][3] These proteins all display a disordered structure and most present a large number of acidic amino acids in their sequence, favoring interactions with crystals [4]. PHEX, an endopeptidase expressed mainly in bone and dentin [16] [17], binds to the ASARM motif within MEPE and prevents its release by CatB [18] [19]. PHEX was shown to bind free ASARM (from MEPE and OPN) as well, neutralize its activity through hydrolysis and abolish its inhibitory action on mineralization [20] [21] [13] [14]. In register with its anti-ASARM properties, PHEX was recently shown to degrade extensively full-length OPN, including the ASARM motif [27] and would be expected to antagonize the inhibitory activity of OPN on mineralization
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