A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype.
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.
Atlanta VA Medical Center and Department of Orthopaedics, Emory University School of Medicine, Atlanta, GA 30329, USA.
Satoshi
S
Kato
Sreedhara
S
Sangadala
Katsuro
K
Tomita
Louisa
L
Titus
Scott D
SD
Boden
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D002460:Cell Line; D054338:Cell Transdifferentiation / Q000187:drug effects*; D004357:Drug Synergism; D004789:Enzyme Activation; D017930:Genes, Reporter; D006801:Humans; D049410:Luciferases, Renilla / Q000096:biosynthesis; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects*; D015675:Osteocalcin / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis; D051785:Smad Proteins / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism; D015854:Up-Regulation
D048868:Adaptor Proteins, Signal Transducing; C480020:Atrnl1 protein, mouse; C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D008565:Membrane Proteins; D011994:Recombinant Proteins; D051785:Smad Proteins; D015675:Osteocalcin; D049410:Luciferases, Renilla; C498342:SMURF1 protein, human; D044767:Ubiquitin-Protein Ligases