title: Strontium attenuates rhBMP-2-induced osteogenic differentiation via formation of Sr-rhBMP-2 complex and suppression of Smad-dependent signaling pathway.
abstract: Strontium (Sr(2+)) has pronounced effects on stimulating bone formation and inhibiting bone resorption in bone regeneration. In this current study, the effect and the underlying mechanism involved of Sr(2+) on the biological activity of bone morphogenetic protein-2 (BMP-2) were studied in detail with pluripotent skeletal muscle myogenic progenitor C2C12 model cell line. The results indicated that Sr(2+) could bind recombinant human BMP-2 (rhBMP-2) rapidly, even in the presence of Ca(2+) and Mg(2+), and inhibited rhBMP-2-induced osteogenic differentiation in vitro and osteogenetic efficiency in vivo. Further studies demonstrated that Sr(2+) treatment undermined the binding capacity of rhBMP-2 with its receptor BMPRIA and thus attenuated Smad 1/5/8 phosphorylation without affecting their dephosphorylation in C2C12 cells. Furthermore, circular dichroism spectroscopy, fluorescence spectroscopy and X-ray photoelectron spectroscopy all revealed that the inhibitory effect of Sr(2+) on the rhBMP-2 osteogenic activity was associated with the formation of Sr-rhBMP-2 complex and ensuing enhancement of β-sheet structure. Our work suggests the activity of rhBMP-2 to induce osteogenic differentiation was decreased by directly interaction with free Sr ions in solution, which should provide guide and assist for development of BMP-2-based materials for bone regeneration.
  
  
  CONCLUSIONS
  Due to easy denaturation and ensuing the reduced activity of rhBMP-2, preserving/enhancing the capacity of rhBMP-2 to induce osteogenic differentiation is of critical importance in developing the protein-based therapy. Cations as effective elements influence the conformation and thereby the bioactivity of protein. Strontium (Sr(2+)), stimulating bone formation and inhibiting bone resorption, has been incorporated into biomaterials/scaffold to improve the bioactivity for bone-regeneration applications. However, Sr(2+)-induced changes in the conformation and bioactivity of BMP-2 have never been investigated. In this study, the formation of Sr-rhBMP-2 complex inhibited the osteogenic differentiation in vitro and osteogenetic efficiency in vivo through the inhibition of BMP/Smad signaling pathway, providing guidance for development of Sr-containing BMP-2-based bone scaffold/matrice and other Sr-dopped protein therapy.
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China; Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Zhang
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China; Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: He
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China; Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, PR China.
authors.forename: Han
authors.initials: H
authors.lastname: Guo
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China; Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China. Electronic address: yyuan@ecust.edu.cn.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China; Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China; Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China. Electronic address: liucs@ecust.edu.cn.
authors.forename: Changsheng
authors.initials: C
authors.lastname: Liu
mesh_terms: D000818:Animals; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000737:chemistry / Q000494:pharmacology*; D052004:Bone Morphogenetic Protein Receptors / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002828:Choristoma / Q000473:pathology; D006801:Humans; D007477:Ions; D051379:Mice; D008954:Models, Biological; D010012:Osteogenesis / Q000187:drug effects*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D051785:Smad Proteins / Q000378:metabolism*; D013324:Strontium / Q000494:pharmacology*; D013816:Thermodynamics; D016212:Transforming Growth Factor beta / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D055396:Bone Morphogenetic Protein 2; D007477:Ions; D011994:Recombinant Proteins; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D052004:Bone Morphogenetic Protein Receptors; D013324:Strontium
keywords: Bone formation; Sr ions; Sr-rhBMP-2 complex; rhBMP-2