Mineralization Osteoblast Differentiation and the Small Molecule Phenamil Induces Supplemental Material

ABSTRACT Stimulation of osteoblast differentiation from mesenchymal stem cells is a potential strategy for bone repair. Bone morphogenetic proteins (BMPs) that induce osteoblastic differentiation have been successfully used in humans to treat fractures. Here we outline a new approach to the stimulation of osteoblast differentiation using small molecules that stimulate BMP activity. We have identified the amiloride derivative phenamil as a stimulator of osteoblast differentiation and mineralization. Remarkably, phenamil acts cooperatively with BMPs to induce the expression of BMP target genes, osteogenic markers, and matrix mineralization in both mesenchymal stem cell lines and calvarial organ cultures. Transcriptional profiling of cells treated with phenamil led to the identification of tribbles homolog 3 (Trb3) as a mediator of its effects. Trb3 is induced by phenamil selectively in cells with osteoblastic potential. Both Trb3 and phenamil stabilize the expression of SMAD, the critical transcription factor in BMP signaling, by promoting the degradation of SMAD ubiquitin regulatory factor 1. Small interfering RNA-mediated knockdown of Trb3 blunts the effects of phenamil on BMP signaling and osteogenesis. Thus, phenamil induces osteogenic differentiation, at least in part, through Trb3-dependent promotion of BMP action. The synergistic use of small molecules such as phenamil along with BMPs may provide new strategies for the promotion of bone healing.

[1]  P. Tontonoz,et al.  Inhibition of adipocyte differentiation by Nur77, Nurr1, and Nor1. , 2008, Molecular endocrinology.

[2]  H. Hayashi,et al.  TRB3 suppresses adipocyte differentiation by negatively regulating PPARgamma transcriptional activity. , 2008, Journal of lipid research.

[3]  F. Parhami,et al.  Oxysterols enhance osteoblast differentiation in vitro and bone healing in vivo , 2007, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[4]  C. Kahn,et al.  TRB3 Blocks Adipocyte Differentiation through the Inhibition of C/EBPβ Transcriptional Activity , 2007, Molecular and Cellular Biology.

[5]  A. Hata,et al.  A Novel Regulatory Mechanism of the Bone Morphogenetic Protein (BMP) Signaling Pathway Involving the Carboxyl-Terminal Tail Domain of BMP Type II Receptor , 2007, Molecular and Cellular Biology.

[6]  S. Nelson,et al.  Oxysterols Are Novel Activators of the Hedgehog Signaling Pathway in Pluripotent Mesenchymal Cells* , 2007, Journal of Biological Chemistry.

[7]  Z. Israili,et al.  Update of Diuretics in the Treatment of Hypertension , 2007, American journal of therapeutics.

[8]  B. Ache,et al.  Block by amiloride derivatives of odor-evoked discharge in lobster olfactory receptor neurons through action on a presumptive TRP channel. , 2006, Chemical senses.

[9]  J. Yates,et al.  TRB3 Links the E3 Ubiquitin Ligase COP1 to Lipid Metabolism , 2006, Science.

[10]  M. Hoshino,et al.  Bone Marrow Stromal Cells Generate Muscle Cells and Repair Muscle Degeneration , 2005, Science.

[11]  H. Kojima,et al.  The Use of Cultured Bone Marrow Cells in Type I Collagen Gel and Porous Hydroxyapatite for Posterolateral Lumbar Spine Fusion , 2005, Spine.

[12]  H. Hayashi,et al.  TRB3, a novel ER stress‐inducible gene, is induced via ATF4–CHOP pathway and is involved in cell death , 2005, The EMBO journal.

[13]  P. Iynedjian.  Lack of evidence for a role of TRB3/NIPK as an inhibitor of PKB-mediated insulin signalling in primary hepatocytes. , 2005, The Biochemical journal.

[14]  D. Ornitz,et al.  Sequential roles of Hedgehog and Wnt signaling in osteoblast development , 2004, Development.

[15]  M. Pittenger,et al.  Mesenchymal stem cells and their potential as cardiac therapeutics. , 2004, Circulation research.

[16]  Mari Dezawa,et al.  Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation. , 2004, The Journal of clinical investigation.

[17]  D. Shouhed,et al.  Oxysterols Regulate Differentiation of Mesenchymal Stem Cells: Pro‐Bone and Anti‐Fat , 2004, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[18]  P. Knaus,et al.  Signal transduction of bone morphogenetic protein receptors. , 2004, Cellular signalling.

[19]  M. Karperien,et al.  Hedgehog stimulates only osteoblastic differentiation of undifferentiated KS483 cells. , 2003, Bone.

[20]  C. Eng,et al.  From developmental disorder to heritable cancer: it's all in the BMP/TGF-β family , 2003, Nature Reviews Genetics.

[21]  K. Miyazono,et al.  Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads. , 2003, Molecular biology of the cell.

[22]  Marc Montminy,et al.  TRB3: A tribbles Homolog That Inhibits Akt/PKB Activation by Insulin in Liver , 2003, Science.

[23]  B. Masereel,et al.  An overview of inhibitors of Na(+)/H(+) exchanger. , 2003, European journal of medicinal chemistry.

[24]  Michael G Fehlings,et al.  Use of osteogenic protein-1 in spinal fusion: literature review and preliminary results in a prospective series of high-risk cases. , 2002, Neurosurgical focus.

[25]  S. Santavirta,et al.  Recombinant Human Bone Morphogenetic Protein-2 for Treatment of Open Tibial Fractures: A Prospective, Controlled, Randomized Study of Four Hundred and Fifty Patients , 2002, The Journal of bone and joint surgery. American volume.

[26]  L. Muul,et al.  Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[27]  G. Sukhikh,et al.  Mesenchymal Stem Cells , 2002, Bulletin of Experimental Biology and Medicine.

[28]  Miikka Vikkula,et al.  LDL Receptor-Related Protein 5 (LRP5) Affects Bone Accrual and Eye Development , 2001, Cell.

[29]  R. Baron,et al.  Sonic hedgehog increases the commitment of pluripotent mesenchymal cells into the osteoblastic lineage and abolishes adipocytic differentiation. , 2001, Journal of cell science.

[30]  C. Sfeir,et al.  Gene therapy approaches for modulating bone regeneration. , 2000, Advanced drug delivery reviews.

[31]  T. Komori,et al.  Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. , 2000, Endocrine reviews.

[32]  P. Rørth,et al.  The level of C/EBP protein is critical for cell migration during Drosophila oogenesis and is tightly controlled by regulated degradation. , 2000, Molecular cell.

[33]  P. Bianco,et al.  Marrow stromal stem cells. , 2000, The Journal of clinical investigation.

[34]  Eric E. Johnson,et al.  Human Bone Morphogenetic Protein Allografting for Reconstruction of Femoral Nonunion , 2000, Clinical orthopaedics and related research.

[35]  Jeffrey L. Wrana,et al.  A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation , 1999, Nature.

[36]  F. Fulfaro,et al.  The role of bisphosphonates in the treatment of painful metastatic bone disease: a review of phase III trials , 1998, Pain.

[37]  D. Prockop.  Marrow Stromal Cells as Stem Cells for Nonhematopoietic Tissues , 1997, Science.

[38]  B. Blazer-Yost,et al.  The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities. , 1997, The American journal of physiology.

[39]  V. Rosen,et al.  The effects of aging on the bone inductive activity of recombinant human bone morphogenetic protein-2. , 1996, Endocrinology.

[40]  C. Kiritsy,et al.  Comparative effects of platelet-derived growth factor-BB and insulin-like growth factor-I, individually and in combination, on periodontal regeneration in Macaca fascicularis. , 1996, Journal of periodontal research.

[41]  J. Wozney,et al.  Transforming Growth Factor-β Gene Family Members and Bone* , 1994 .

[42]  J N Beresford,et al.  Osteogenic stem cells and the stromal system of bone and marrow. , 1989, Clinical orthopaedics and related research.

[43]  E. Cragoe,et al.  Amiloride and its analogs as tools in the study of ion transport , 1988, The Journal of Membrane Biology.

[44]  M. Lazdunski,et al.  Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel, the Na+/H+ antiport and the Na+/Ca2+ exchanger. , 1988, Biochimie.

[45]  D. Benos,et al.  Amiloride: a molecular probe of sodium transport in tissues and cells. , 1982, The American journal of physiology.

[46]  D. Feldman,et al.  A receptor-like binding macromolecule for 1 alpha, 25-dihydroxycholecalciferol in cultured mouse bone cells. , 1979, The Journal of biological chemistry.

[47]  G. Wilson,et al.  Clinical evaluation of amiloride, a potassium‐sparing diuretic , 1969, Clinical pharmacology and therapeutics.

[48]  Charles C Hong,et al.  Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. , 2008, Nature chemical biology.

[49]  In Ho Choi,et al.  A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva , 2006, Nature Genetics.

[50]  Marie-Pierre Dubé,et al.  Mutations in HFE2 cause iron overload in chromosome 1q–linked juvenile hemochromatosis , 2004, Nature Genetics.

[51]  C. Eng,et al.  From developmental disorder to heritable cancer: it's all in the BMP/TGF-beta family. , 2003, Nature reviews. Genetics.

[52]  T. Appelboom,et al.  Analgesic Effect of Intravenous Pamidronate on Chronic Back Pain due to Osteoporotic Vertebral Fractures , 1999, Clinical Rheumatology.

[53]  J. Wozney,et al.  Transforming growth factor-beta gene family members and bone. , 1994, Endocrine reviews.