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.

The extracellular regulation of bone morphogenetic protein signaling

“…The solidus between names indicates alternate names or homologs in different species. a Reviewed by Garcia Abreu et al, 2002; reviewed by Gazzerro and Canalis, 2006; c reviewed by Balemans and Van Hul, 2002;d Vilmos et al, 2001;e Cowan et al, 2007;f Yanagita, 2005;g Yu et al, 2008;h Paine-Saunders et al, 2000;i Fujise et al, 2001;j Kirkpatrick et al, 2006;k Gumienny et al, 2007;l Fisher et al, 2006;m Moreno et al, 2005;n Ohta et al, 2004; o Kirkbride et al, 2008;p Fung et al, 2007;q Wilkinson et al, 2003;r Sieron et al, 2002;s Zhu et al, 1999;t Moser et al, 2003;u Serpe et al, 2008;v Lin et al, 2005;w Matsui et al, 2000;x Babitt et al, 2005;z Kanomata et al, 2009;aa Onichtchouk et al, 1999;bb Yao et al, 2008;cc Wallin et al, 2000;dd Brochmann et al, 2009;ee Behnam et al, 2006;ff Rittenberg et al, 2005;gg Szweras et al, 2002;hh Saito et al, 2008; ii Wang et al, 2008;jj Arteaga-Solis et al, 2001; kk Sengle et al, 2008a. REVIEW Development 136 (22) degradation of BMP-bound receptors; at present, no extracellular proteases that specifically target ligands of the transforming growth factor  (TGF) superfamily for destruction have ...…”

Dermatopontin in the extracellular matrix enhances osteogenic differentiation of adipose-derived mesenchymal stem cells

“…Most convincingly, DPT modulates cell behavior indirectly by modulating cell-ECM interactions [1], DPT binds several known growth factors including TGFβ and BMP2 [3,4], altered DPT expression is observed during tissue injury [28][29][30][31][32][33], and DPT knockout mice only show subtle changes to skin elasticity [7]. In addition, Okamoto & Fujiwara suggest that DPT contributes predominately to matrix remodeling during wound healing and that further studies on wound healing in DPT transgenic mice mayeluci date the specific roles of DPT in response to injury [1].…”

“…Dermatopontin (DPT) is a 22-kDa tyrosine-rich extracellular matrix (ECM) protein that is found at high levels in demineralized bone matrix and cartilage and may play an important role in skeletal tissue function [1][2][3]. It is thought that DPT acts largely as a mediator between the cell and the ECM environment [1,4,5].…”

“…It is thought that DPT acts largely as a mediator between the cell and the ECM environment [1,4,5]. Functional roles attributed to DPT with relevance to bone include binding and modulation of bone morphogenetic protein 2 (BMP2) and transforming growth factor b (TGFb), cell adhesion, collagen and fibronectin fibril formation, cell proliferation, and reported activity as a downstream target of the vitamin D receptor (VDR) [1,3,4,[6][7][8][9].…”

The Use of Mass Spectrometry in Characterization of Bone Morphogenetic Proteins from Biological Samples

“…Using LC-MS/MS authors identified this factor as BMP4, and found it to be in over 100 kDa complex in circulation. Behnam et al (2006) identified dermatopontin (DPT) as the most prominent BMP-2 co-purified demineralized bone matrix protein, using alkaline urea extraction of BM, separation of insoluble fraction by hydroxyapatite chromatography, SDS-PAGE of BMP active fraction and MALDI-TOF analysis. Kubota et al (2002) have studied signal transduction from osteoclasts to osteoblasts.…”