Retinoic Acids Potentiate BMP9-Induced Osteogenic Differentiation of Mesenchymal Progenitor Cells

Background As one of the least studied bone morphogenetic proteins (BMPs), BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA) signaling is known to play an important role in development, differentiation and bone metabolism. In this study, we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs). Methodology/Principal Findings Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9, RARα and RXRα into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as osteopontin (OPN) and osteocalcin (OC). BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants, we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRα or RARα significantly increases trabecular bone and osteoid matrix formation. Conclusion/Significance Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore, a combination of BMP9 and retinoic acid (or its agonists) may be explored as effective bone regeneration therapeutics to treat large segmental bony defects, non-union fracture, and/or osteoporotic fracture.

[1]  T. He,et al.  Synergistic Antitumor Effect of the Activated PPARγ and Retinoid Receptors on Human Osteosarcoma , 2010, Clinical Cancer Research.

[2]  T. He,et al.  Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells. , 2009, Differentiation; research in biological diversity.

[3]  T. He,et al.  Retinoic acid signalling induces the differentiation of mouse fetal liver‐derived hepatic progenitor cells , 2009, Liver international : official journal of the International Association for the Study of the Liver.

[4]  T. He,et al.  Wnt antagonist SFRP3 inhibits the differentiation of mouse hepatic progenitor cells , 2009, Journal of cellular biochemistry.

[5]  T. He,et al.  BMP‐9‐induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/β‐catenin signalling , 2009, Journal of cellular and molecular medicine.

[6]  T. He,et al.  A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells. , 2009, Stem cells and development.

[7]  T. He,et al.  Hey1 Basic Helix-Loop-Helix Protein Plays an Important Role in Mediating BMP9-induced Osteogenic Differentiation of Mesenchymal Progenitor Cells* , 2009, Journal of Biological Chemistry.

[8]  A. Montag,et al.  Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects , 2008, Laboratory Investigation.

[9]  Anxun Wang,et al.  Retinoic acid inhibits osteogenic differentiation of rat bone marrow stromal cells. , 2008, Biochemical and biophysical research communications.

[10]  G. Duester.  Retinoic Acid Synthesis and Signaling during Early Organogenesis , 2008, Cell.

[11]  T. He,et al.  Osteosarcoma Development and Stem Cell Differentiation , 2008, Clinical orthopaedics and related research.

[12]  P. Dollé,et al.  Retinoic acid in development: towards an integrated view , 2008, Nature Reviews Genetics.

[13]  K. Zaret.  Genetic programming of liver and pancreas progenitors: lessons for stem-cell differentiation , 2008, Nature Reviews Genetics.

[14]  C. Kaps,et al.  Human mastoid periosteum‐derived stem cells: promising candidates for skeletal tissue engineering , 2008, Journal of tissue engineering and regenerative medicine.

[15]  B. Sacchetti,et al.  Self-Renewing Osteoprogenitors in Bone Marrow Sinusoids Can Organize a Hematopoietic Microenvironment , 2007, Cell.

[16]  J. Berger,et al.  Retinaldehyde represses adipogenesis and diet-induced obesity , 2007, Nature Medicine.

[17]  A. Montag,et al.  Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells , 2007, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[18]  K. Kinzler,et al.  A protocol for rapid generation of recombinant adenoviruses using the AdEasy system , 2007, Nature Protocols.

[19]  Luisa Lassová,et al.  Retinoids directly activate the collagen X promoter in prehypertrophic chondrocytes through a distal retinoic acid response element , 2006, Journal of cellular biochemistry.

[20]  M. Longaker,et al.  Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling , 2006, Proceedings of the National Academy of Sciences.

[21]  E. Beutler,et al.  Bone morphogenetic proteins 2, 4, and 9 stimulate murine hepcidin 1 expression independently of Hfe, transferrin receptor 2 (Tfr2), and IL-6 , 2006, Proceedings of the National Academy of Sciences.

[22]  Kamal Garcha,et al.  BMP action in skeletogenesis involves attenuation of retinoid signaling , 2006, The Journal of cell biology.

[23]  Lindolfo da Silva Meirelles,et al.  Mesenchymal stem cells reside in virtually all post-natal organs and tissues , 2006, Journal of Cell Science.

[24]  A. Montag,et al.  CCN1/Cyr61 Is Regulated by the Canonical Wnt Signal and Plays an Important Role in Wnt3A-Induced Osteoblast Differentiation of Mesenchymal Stem Cells , 2006, Molecular and Cellular Biology.

[25]  P. Chambon,et al.  Function of retinoid nuclear receptors: lessons from genetic and pharmacological dissections of the retinoic acid signaling pathway during mouse embryogenesis. , 2006, Annual review of pharmacology and toxicology.

[26]  Robert M Hoffman,et al.  Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[27]  J. Wrana,et al.  The disparate role of BMP in stem cell biology , 2005, Oncogene.

[28]  Jiwang Zhang,et al.  BMP signaling and stem cell regulation. , 2005, Developmental biology.

[29]  Benjamin M. Wu,et al.  Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover. , 2005, Tissue engineering.

[30]  A. Montag,et al.  Connective Tissue Growth Factor (CTGF) Is Regulated by Wnt and Bone Morphogenetic Proteins Signaling in Osteoblast Differentiation of Mesenchymal Stem Cells* , 2004, Journal of Biological Chemistry.

[31]  F. Claas,et al.  Isolation of Mesenchymal Stem Cells of Fetal or Maternal Origin from Human Placenta , 2004, Stem cells.

[32]  D. van der Kooy,et al.  Facile isolation and the characterization of human retinal stem cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[33]  L. Martin,et al.  Pluripotent fates and tissue regenerative potential of adult olfactory bulb neural stem and progenitor cells. , 2004, Journal of neurotrauma.

[34]  J. Szatkowski,et al.  Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery , 2004, Gene Therapy.

[35]  Mark P. Lewis,et al.  Human adult craniofacial muscle‐derived cells: neural‐cell adhesion‐molecule (NCAM; CD56)‐expressing cells appear to contain multipotential stem cells , 2004, Biotechnology and applied biochemistry.

[36]  Wei Jiang,et al.  Inhibitor of DNA Binding/Differentiation Helix-Loop-Helix Proteins Mediate Bone Morphogenetic Protein-induced Osteoblast Differentiation of Mesenchymal Stem Cells* , 2004, Journal of Biological Chemistry.

[37]  A. Matsuda,et al.  Homeobox Protein Msx2 Acts as a Molecular Defense Mechanism for Preventing Ossification in Ligament Fibroblasts , 2004, Molecular and Cellular Biology.

[38]  E. Garattini,et al.  Retinoid related molecules an emerging class of apoptotic agents with promising therapeutic potential in oncology: pharmacological activity and mechanisms of action. , 2004, Current pharmaceutical design.

[39]  Hongwei Cheng,et al.  Transcriptional characterization of bone morphogenetic proteins (BMPs)‐mediated osteogenic signaling , 2003, Journal of cellular biochemistry.

[40]  E. Schwarz,et al.  Runx2/Cbfa1 stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smad1 on the collagen X promoter in chondrocytes , 2003, Journal of cellular biochemistry.

[41]  E. Dmitrovsky,et al.  Retinoids in cancer therapy and chemoprevention: promise meets resistance , 2003, Oncogene.

[42]  Di Chen,et al.  E3 Ubiquitin Ligase Smurf1 Mediates Core-binding Factor α1/Runx2 Degradation and Plays A Specific Role in Osteoblast Differentiation* , 2003, Journal of Biological Chemistry.

[43]  J. Massagué,et al.  Mechanisms of TGF-β Signaling from Cell Membrane to the Nucleus , 2003, Cell.

[44]  E. Schwarz,et al.  Retinoic acid stimulates chondrocyte differentiation and enhances bone morphogenetic protein effects through induction of Smad1 and Smad5. , 2003, Endocrinology.

[45]  Mallika Singh,et al.  An integrated functional genomics screening program reveals a role for BMP-9 in glucose homeostasis , 2003, Nature Biotechnology.

[46]  Guang-Quan Zhao,et al.  Consequences of knocking out BMP signaling in the mouse , 2003, Genesis.

[47]  R. Derynck,et al.  Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes , 2002, The Journal of cell biology.

[48]  Jeffrey L. Wrana,et al.  Signal Transduction by the TGF-β Superfamily , 2002, Science.

[49]  A. Montag,et al.  Nuclear receptor agonists as potential differentiation therapy agents for human osteosarcoma. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[50]  R. Haselbeck,et al.  Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice. , 2002, Development.

[51]  H. Gronemeyer,et al.  Co-regulator recruitment and the mechanism of retinoic acid receptor synergy , 2002, Nature.

[52]  R. Evans,et al.  Nuclear receptors and lipid physiology: opening the X-files. , 2001, Science.

[53]  H. Lorenz,et al.  Multilineage cells from human adipose tissue: implications for cell-based therapies. , 2001, Tissue engineering.

[54]  B. Park,et al.  Peroxisome proliferator-activated receptors: roles in tumorigenesis and chemoprevention in human cancer , 2001, Current opinion in oncology.

[55]  J. Holder,et al.  PPAR-γ agonists: therapeutic role in diabetes, inflammation and cancer , 2000 .

[56]  J. Blusztajn,et al.  Induction and maintenance of the neuronal cholinergic phenotype in the central nervous system by BMP-9. , 2000, Science.

[57]  Sander Kersten,et al.  Roles of PPARs in health and disease , 2000, Nature.

[58]  Vicki Rosen,et al.  Regulation of Skeletal Progenitor Differentiation by the Bmp and Retinoid Signaling Pathways , 2000, The Journal of cell biology.

[59]  A. Wolffe,et al.  Nuclear receptors: coactivators, corepressors and chromatin remodeling in the control of transcription. , 1999, Journal of molecular endocrinology.

[60]  K. Kinzler,et al.  PPARδ Is an APC-Regulated Target of Nonsteroidal Anti-Inflammatory Drugs , 1999, Cell.

[61]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.

[62]  L. Cook,et al.  Modulation of bone morphogenetic protein-2 and bone morphogenetic protein-4 gene expression in osteoblastic cell lines. , 1998, Cellular and molecular biology.

[63]  A. Sparks,et al.  Identification of c-MYC as a target of the APC pathway. , 1998, Science.

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

[65]  K. Schughart,et al.  Retinoic Acid Receptor α Function in Vertebrate Limb Skeletogenesis: a Modulator of Chondrogenesis , 1997, The Journal of cell biology.

[66]  B. Hogan,et al.  Bone morphogenetic proteins: multifunctional regulators of vertebrate development. , 1996, Genes & development.

[67]  S. Hatakeyama,et al.  Retinoic acid enhances expression of bone morphogenetic protein-2 in human adenocarcinoma cell line (HSG-S8). , 1996, Biochemistry and molecular biology international.

[68]  K. Umesono,et al.  The nuclear receptor superfamily: The second decade , 1995, Cell.

[69]  V. Rosen,et al.  Bone morphogenetic protein-9 binds to liver cells and stimulates proliferation. , 1995, Endocrinology.

[70]  P. Chambon,et al.  Targeted disruption of retinoic acid receptor alpha (RAR alpha) and RAR gamma results in receptor-specific alterations in retinoic acid-mediated differentiation and retinoic acid metabolism , 1995, Molecular and cellular biology.

[71]  S. Temple,et al.  A self-renewing multipotential stem cell in embryonic rat cerebral cortex , 1994, Nature.

[72]  V. Rosen,et al.  Bone morphogenetic proteins-2 and -4 are involved in the retinoic acid-induced differentiation of embryonal carcinoma cells. , 1992, Molecular biology of the cell.

[73]  A. Montag,et al.  Regulation of osteogenic differentiation during skeletal development. , 2008, Frontiers in bioscience : a journal and virtual library.

[74]  M. Bjerknes,et al.  Intestinal epithelial stem cells and progenitors. , 2006, Methods in enzymology.

[75]  J. Massagué,et al.  Mechanisms of TGF-beta signaling from cell membrane to the nucleus. , 2003, Cell.

[76]  J. Szatkowski,et al.  Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). , 2003, The Journal of bone and joint surgery. American volume.

[77]  J. Wrana,et al.  Signal transduction by the TGF-beta superfamily. , 2002, Science.

[78]  B. Olsen,et al.  Bone development. , 2000, Annual review of cell and developmental biology.

[79]  J. Holder,et al.  PPAR-gamma agonists: therapeutic role in diabetes, inflammation and cancer. , 2000, Trends in pharmacological sciences.

[80]  K. Kinzler,et al.  A simplified system for generating recombinant adenoviruses. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[81]  J. Aubin,et al.  Advances in the osteoblast lineage. , 1998, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[82]  K. Schughart,et al.  Retinoic Acid Receptor (cid:97) Function in Vertebrate Limb Skeletogenesis: a Modulator of Chondrogenesis , 1997 .