title: BMP stimulation of alkaline phosphatase activity in pluripotent mouse C2C12 cells is inhibited by dermatopontin, one of the most abundant low molecular weight proteins in demineralized bone matrix.
abstract: Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl2, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of gamma-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.
authors.affiliation: Department of Physiological Sciences, University of California, Los Angeles, CA, USA.
authors.forename: Keyvan
authors.initials: K
authors.lastname: Behnam
authors.forename: Samuel S
authors.initials: SS
authors.lastname: Murray
authors.forename: Elsa J
authors.initials: EJ
authors.lastname: Brochmann
mesh_terms: D000469:Alkaline Phosphatase / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001857:Bone Matrix / Q000187:drug effects / Q000378:metabolism*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis / Q000494:pharmacology / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000494:pharmacology / Q000502:physiology*; D006131:Growth Inhibitors / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D006657:Histones / Q000032:analysis / Q000378:metabolism; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D040901:Proteomics; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D011508:Chondroitin Sulfate Proteoglycans; C504453:Dpt protein, mouse; D016326:Extracellular Matrix Proteins; D006131:Growth Inhibitors; D006657:Histones; D016212:Transforming Growth Factor beta; D000469:Alkaline Phosphatase