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A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype

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Abstract

There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1−/− knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored.

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Abbreviations

BMP:

Bone morphogenetic protein

Smurf1:

Smad ubiquitin regulatory factor-1

RT-PCR:

Reverse transcriptase-polymerase chain reaction

ALP:

Alkaline phosphatase

DMSO:

Dimethylsulfoxide

RUL:

Relative units of luciferase

FBS:

Fetal bovine serum

CAP database:

Chemicals available for purchase database

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Disclosures

All the biochemical studies in this work were performed at the Atlanta Veterans Affairs Medical Center and partly supported by NIH grant # R01 AR53093 (Boden) and a VA Merit award to Dr. Titus. In the past and not related to this work, Dr. Boden has received compensation as a consultant for Medtronic Sofamor Danek and for intellectual property. Emory University and some of the authors have/may receive royalties in the future related to LMP-1. The terms of this arrangement have been reviewed and approved by Emory University in accordance with its conflict of interest policies. We thank Boojala VB Reddy for assistance in homology modeling. We also thank Vandana Voleti for assistance in computational analyses.

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Authors and Affiliations

  1. Atlanta VA Medical Center and Department of Orthopaedics, Emory University School of Medicine, Atlanta, GA, 30329, USA

    Satoshi Kato, Sreedhara Sangadala, Louisa Titus & Scott D. Boden

  2. Department of Orthopaedic Surgery, Kanazawa University School of Medicine, Kanazawa, 920-8641, Japan

    Satoshi Kato & Katsuro Tomita

  3. VAMC-Research Service, 1670 Clairmont Rd., Decatur, GA, 30033, USA

    Sreedhara Sangadala

Authors
  1. Satoshi Kato
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  2. Sreedhara Sangadala
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  3. Katsuro Tomita
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  4. Louisa Titus
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  5. Scott D. Boden
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Corresponding author

Correspondence to Sreedhara Sangadala.

Additional information

Satoshi Kato and Sreedhara Sangadala contributed equally to this work.

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Kato, S., Sangadala, S., Tomita, K. et al. A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype. Mol Cell Biochem 349, 97–106 (2011). https://doi.org/10.1007/s11010-010-0664-6

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  • DOI: https://doi.org/10.1007/s11010-010-0664-6

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