title: 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-microm 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 osteoblast-derived 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.
authors.affiliation: Bone Biology Program, Department of Oral Biology, School of Dentistry, University of Missouri, Kansas City, Missouri 64108, USA.
authors.forename: Nichole T
authors.initials: NT
authors.lastname: Huffman
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Keightley
authors.forename: Cui
authors.initials: C
authors.lastname: Chaoying
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Midura
authors.forename: Dinah
authors.initials: D
authors.lastname: Lovitch
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Veno
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Dallas
authors.forename: Jeff P
authors.initials: JP
authors.lastname: Gorski
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002113:Calcification, Physiologic / Q000187:drug effects / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D006023:Glycoproteins / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D053495:Osteopontin / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012886:Skull / Q000166:cytology / Q000378:metabolism*; D013450:Sulfones / Q000494:pharmacology
chemical_list: D015415:Biomarkers; D006023:Glycoproteins; D015842:Serine Proteinase Inhibitors; C506896:Spp1 protein, mouse; D013450:Sulfones; D053495:Osteopontin; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D012697:Serine Endopeptidases