Association of Specific Proteolytic Processing of Bone Sialoprotein and Bone Acidic Glycoprotein-75 with Mineralization within Biomineralization Foci

Abstract

Mineral crystal nucleation in UMR 106-01 osteoblastic cultures occurs within 15-25-m extracellular vesicle-containing biomineralization foci (BMF) structures. We show here that BAG-75 and BSP, biomarkers for these foci, are specifically enriched in laser capture microscope-isolated mineralized BMF as compared with the total cell layer. Unexpectedly, fragments of each protein (45-50 kDa in apparent size) were also enriched within captured BMF. When a series of inhibitors against different protease classes were screened, serine protease inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride HCl (AEBSF) was the only one that completely blocked mineral nucleation within BMF in UMR cultures. AEBSF appeared to act on an osteoblastderived protease at a late differentiation stage in this culture model just prior to mineral deposition. Similarly, mineralization of bone nodules in primary mouse calvarial osteoblastic cultures was completely blocked by AEBSF. Cleavage of BAG-75 and BSP was also inhibited at the minimum dosage of AEBSF sufficient to completely block mineralization of BMF. Two-dimensional SDS-PAGE comparisons of AEBSF-treated and untreated UMR cultures showed that fragmentation/activation of a limited number of other mineralization-related proteins was also blocked. Taken together, our results indicate for the first time that cleavage of BAG-75 and BSP by an AEBSF-sensitive, osteoblast-derived serine protease is associated with mineral crystal nucleation in BMF and suggest that such proteolytic events are a permissive step for mineralization to proceed.Bone is a vascularized tissue that uniquely becomes mineralized as part of its developmental program (1). Mineralized bone serves essential vertebrate functions, including structural support, reversible storage for calcium and phosphorus, and as a reservoir for toxic metals and carbonate (2). Bone tissue is composed of osteoid; osteoblasts, which produce and mineralize new bone; osteoclasts, which resorb bone; and osteocytes, mature osteoblasts that maintain bone viability (1-3). Osteoid is a type I collagen-rich extracellular matrix enriched in acidic noncollagenous proteins (4). Using fetal rat calvaria cell cultures, Bellows et al. (5) showed that osteoid is unmineralized when initially deposited, and mineral crystals form within nodular structures over the following 48 -72 h. Bone matrices can be classified as lamellar, based on a highly organized layered structure, or woven bone. Woven bone is formed during embryonic development, fracture healing, and at sites receiving mechanical stimulation in excess of 3,000 microstrain (6); lamellar bone replaces woven bone later in development.The question of whether bone mineralization is under direct osteoblastic control or whether it is purely a passive chemical process is under active investigation. Schinke et al. (7) have proposed that calcification reactions in vivo are passive physiochemical processes occurring readily where local mineralization inhibitors are overwhelmed. In support of this hypothesis, Murshed et ...

Human Serine Protease HTRA1 Positively Regulates Osteogenesis of Human Bone Marrow-derived Mesenchymal Stem Cells and Mineralization of Differentiating Bone-forming Cells Through the Modulation of Extracellular Matrix Protein

“…The primary biological function of IBSP is unclear, although it has been reported to be involved in the initiation of matrix mineralization through the nucleation of hydroxyapatite crystals [40,41]. Furthermore, it has been proposed that IBSP is required to undergo fragmentation, possibly through the actions of serine proteases, in order to allow for efficient matrix mineralization [15,42,43]. In this report, we were able to demonstrate colocalization of HTRA1 and IBSP in osteo-induced mASC spheroid cultures, thus confirming the potential for endogenous HTRA1 to interact with IBSP under osteogenic culture conditions.…”

“…Of the osteogenic genes that were regulated by HTRA1, IBSP held particular relevance to this study due to the fact that its ability to regulate mineral deposition has previously been reported to be dependent on the actions of serine proteases [15]. Attention was therefore focused on IBSP and its potential involvement in mediating the effects of HTRA1 on both osteogenesis of MSCs and mineralization of differentiating bone-forming cells.…”

“…A direct interaction between these proteins and bone progenitor cells is essential for the development of a mineralized matrix [12][13][14]. Furthermore, degradation of specific glycoproteins within the ECM by serine proteases is also thought to play a central role in controlling mineral deposition by osteoblasts [15].…”

Clinical Concepts in Oral and Maxillofacial Surgery and Novel Findings to the Field of Bone Regeneration

“…Matrix vesicles have long been contested as sites of nucleation in bone and recent data suggested that the vesicles are present in bone but that they vary in size and the composition (Gorski, 2011). Vesicles have also been shown to be present within biomineralization foci (Huffman et al, 2007). Biomineralization foci (10-25 micron diameter) are the result of ECM-mediated nucleation.…”

Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age

“…In terms of protein, osteocyte-enriched control bone extracts contained an 18-kDa MBTPS1 fragment band also enriched in laser microscope-dissected biomineralization foci (Fig. 1a), sites of initial deposition of hydroxyapatite in osteoblastic cultures (29).…”