Full‐length bovine spp24 [spp24 (24‐203)] inhibits BMP‐2 induced bone formation

Abstract

Secreted phosphoprotein 24 kDa (spp24) is a bone matrix protein. It contains a TGF-b receptor II homology 1 (TRH1) domain. A cyclic, synthetic 19 amino acid peptide (bone morphogenetic protein binding peptide or BBP) based on the sequence of the TRH1 domain enhances BMP-2 induced osteogenesis. Many observations suggest that different size forms of this protein have very different effects (inhibiting or enhancing) on BMP-2 induced osteogenesis. Using the stable recombinant Met(His) 6 -tagged secretory form of full-length (fl) bovine spp24 [Met(His) 6 -spp24 (residues 24-203)] and transgenic (TG) mice expressing fl bovine spp24 (residues 1-203), we have demonstrated that spp24 inhibits BMP-2 induced bone formation. The effects of Met(His) 6 -spp24 (24-203) were determined in the ectopic bone-forming bioassay in male mice. Implantation of 5 mg of BMP-2 stimulated bone formation, assessed densitometrically as bone area and mineral content. When Met(His) 6 -spp24 (24-203) was implanted with BMP-2, it elicited a dose-dependent decrease in BMP-2-medicated ectopic bone formation. When added at a 50-fold excess (w/w), Met(His) 6 -spp24 (24-203) completely ablated the effects of BMP-2, while addition of a 10-fold excess had no effect. Constitutive expression of fl bovine spp24 (1-203) under the control of the osteocalcin promoter in TG female mice reduced femoral and vertebral bone mineral density at 3 months of age and reduced femoral BMD at 8 months of age, but had no effects in male mice, which can exhibit less osteocalcin-promoter driven gene transcription than females. Histomorphometric analysis demonstrated that bone volume and trabecular thickness were lower in TG female mice at 3 months of age than in sex-and age-matched wild type (WT) controls. Thus, fl spp24 and its secretory isoform (Met(His) 6 -spp24 ), which contain a BMP-binding or TRH1 motif, inhibit ectopic bone formation in male mice and adversely affects BMD and histological parameters related to bone mass and formation in female mice expressing the human transgene. Under these conditions, fl spp24 acts as a BMP antagonist in vivo. Keywords: bone formation; spp24; bone morphogenetic protein Secreted phosphoprotein-24 (spp24) is a 24 kDa protein of unknown function first cloned from bovine bone matrix.1 The calculated molecular weight of the fulllength unprocessed precursor [bovine spp24 (residues 1-203)] is 23.1 kDa (Swiss-Prot database; AC #Q27967). The first 23 amino acid residues of bovine spp24 (1-203) constitute a signal peptide that is cleaved when the protein is secreted in its mature form [spp24 (24-203)]. The protein contains two internal disulfide bonds linking residues 86 to 97 and 110 to 128, respectively, and contains internal domains with sequence homology to cystatins (cysteine protease inhibitors) and cathelicidins (protease-sensitive precursors of endogenous antibiotic peptides).1 Recent work demonstrated that an 18.5 kDa protein in the osteogenic fraction that Urist et al.2 isolated from demineralized bone matrix (DBM) by hy...

Carboxy terminus of secreted phosphoprotein‐24 kDa (spp24) is essential for full inhibition of BMP‐2 activity

“…15,16 It binds hydroxyapatite, 6,17 stimulates cathepsins, 18 and binds BMP-2 (Table 1) as well as BMP-7, GDF-5, and TGF-b (unpublished observations). Native spp24 inhibits bone formation, 8 whereas a 19 amino acid peptide based on the sequence of the BMP-binding region has been developed as a bone healing agent presumably based on a ''slow release'' (of BMP-2) mechanism. 6 We have investigated the BMP-2 binding properties and the effect on BMP bone forming activity of four forms of spp24 which we contend represent the major proteolytic products found in bone (spp18.1) 6 or which represent cleavages at exquisitely labile sites in the recombinant protein (spp14.5 and spp16).…”

“…For example, implantation of recombinant fl-spp24 (24-203) inhibits ectopic bone formation, and overexpression of spp24 is associated with reduced vertebral and femoral bone mineral density, cancellous bone volume, and trabecular thickness and increased trabecular separation in 3-month-old female transgenic mice. 8 In contrast, native bone spp18.5 enhances bone formation, 3,4,19 and BBP (BMP binding peptide; the BMP-2-binding 19-amino acid residue cyclic peptide corresponding to residues 110 to 128 of bovine spp24) enhances BMP-2-mediated ectopic bone formation 6 and spinal fusion. 7 We hypothesized that proteolytic degradation of spp24 modulates its bioactivity, assessed as BMP-2 binding, or the effects on BMP-2-mediated ectopic bone formation.…”

“…A synthetic cyclic peptide (bone morphogenetic binding peptide, BBP) based on that sequence was shown to bind rhBMP-2 and enhance the bone forming activity of BMP-2. 6,7 On the other hand, full-length spp24 was shown to inhibit bone formation at ectopic sites, 8 in transgenic animals, 8 and in a rodent model of spinal fusion (manuscript in preparation). Thus, proteolytic processing converts spp24 from an inhibitor of bone formation 8 to an 18.5 kDa proosteogenic protein.…”

“…6,7 On the other hand, full-length spp24 was shown to inhibit bone formation at ectopic sites, 8 in transgenic animals, 8 and in a rodent model of spinal fusion (manuscript in preparation). Thus, proteolytic processing converts spp24 from an inhibitor of bone formation 8 to an 18.5 kDa proosteogenic protein. 3,4 The purposes of the current studies were to initiate an understanding of the function of spp24 in vivo and to evaluate the structure/function relationship of various spp24 size forms in a manner that would assist in the rational development of bone healing therapeutics.…”

Bone morphogenetic protein–2 activity is regulated by secreted phosphoprotein–24 kd, an extracellular pseudoreceptor, the gene for which maps to a region of the human genome important for bone quality

“…Alternatively, the acidic residues of the cystatin domain of spp24 may form a negatively charged planar β-sheet that inhibits basic calcium phosphate precipitation in a manner similar to that observed for the D1 region of the structurally similar cystatin domain of fetuin [42]. This could explain, in part, the observations that overexpression of full-length (fl) b-spp24 inhibits murine bone formation in vivo and that implantation of recombinant His 6 -tagged secretory b-spp24 (residues 24-203) dose-dependently inhibits BMP-2-mediated ectopic bone formation [43].…”