title: A novel library screen identifies immunosuppressors that promote osteoblast differentiation.
abstract: Bone homeostasis can be compromised by an increase in osteoclast-mediated resorption and/or a decrease in osteoblast-mediated bone deposition. While many efforts have focused on treating osteoclast resorption, there has been less emphasis on identifying strategies for promoting osteoblast function. Herein, we describe a high-throughput screening assay to select for small molecules that augment bone morphogenetic protein-2 (BMP-2)-mediated osteoblast lineage commitment. After an initial screen of 5405 compounds; consisting of FDA-approved drugs, known bioactives, and compounds with novel chemical makeup, we identified 45 small molecules that promoted osteoblast commitment. Of the 45 candidates, there was a broad array of classes that included nine retinoid analogs/derivatives and four immunosuppressants, notably rapamycin and FK-506, which were chosen for further study. Treatment of osteoblast precursor cells with rapamycin or FK-506, either alone, or synergistically with BMP-2, increased levels of phospho-Smad 1/5/8 protein and transcription of Runx-2, Osx and Smad-7, consistent with a role in promoting osteoblast differentiation. Only FK-506 was able to enhance osteocalcin transcripts and Alizarin Red staining, both late markers for differentiation. When osteoblast differentiation was suppressed with exogenous TGF-β1 treatment, rapamycin (but not FK-506) was able to rescue expression of differentiation markers, indicating distinct but overlapping activity of these compounds. Collectively, these data add to an understanding of pathways engaged in osteoblastogenesis, support a role for non-redundant immunosuppressant signaling, and provide a novel approach for the discovery of potentially therapeutic compounds that affect bone remodeling.
authors.affiliation: Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Ariana
authors.initials: A
authors.lastname: Darcy
authors.forename: Micah
authors.initials: M
authors.lastname: Meltzer
authors.forename: Joseph
authors.initials: J
authors.lastname: Miller
authors.forename: Steven
authors.initials: S
authors.lastname: Lee
authors.forename: Scott
authors.initials: S
authors.lastname: Chappell
authors.forename: Kris
authors.initials: K
authors.lastname: Ver Donck
authors.forename: Monty
authors.initials: M
authors.lastname: Montano
mesh_terms: D016475:3T3 Cells; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000008:administration & dosage / Q000494:pharmacology; D016723:Bone Remodeling / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004357:Drug Synergism; D057166:High-Throughput Screening Assays / Q000379:methods*; D007166:Immunosuppressive Agents / Q000008:administration & dosage / Q000494:pharmacology*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000008:administration & dosage / Q000494:pharmacology; D051906:Smad7 Protein / Q000235:genetics; D054852:Small Molecule Libraries / Q000008:administration & dosage / Q000494:pharmacology*; D000076182:Sp7 Transcription Factor; D016559:Tacrolimus / Q000008:administration & dosage / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics; D053773:Transforming Growth Factor beta1 / Q000008:administration & dosage
chemical_list: C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; D007166:Immunosuppressive Agents; D012333:RNA, Messenger; C493716:Runx2 protein, mouse; D051906:Smad7 Protein; C497807:Smad7 protein, mouse; D054852:Small Molecule Libraries; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D014157:Transcription Factors; D053773:Transforming Growth Factor beta1; D020123:Sirolimus; D016559:Tacrolimus