The promise of skeletal regenerative medicine to mitigate age-related bone loss and support fracture healing critically depends on the ability to manipulate the biological properties of osteogenic progenitor cells. Transcriptional and post-transcriptional regulatory mechanisms control cell fate determination and phenotype- specific gene expression when progenitor cells commit to the osteoblast lineage. MicroRNAs (miRNAs or miRs) and epigenetic regulators (EpiRegs) regulate osteogenic differentiation by controlling the expression of TFs. Therefore, this proposal will focus on miR-TF-EpiReg circuits that control osteoblast differentiation. The central hypothesis of the overall project is that osteoblast differentiation is controlled by miRNAs- TF-EpiReg circuits. Based on recent preliminary data, this proposal has evolved to address the specific working model that miR155 suppresses while miR101 stimulates osteoblast maturation by targeting distinct TFs and EpiRegs, respectively. Maximal expression of miR155 occurs in undifferentiated osteoblasts where we predict that miR155 - together with co-regulated miRNAs - controls a group of common TF targets that attenuate osteoblast maturation. For miR101, we observed that it is maximal in mature osteoblasts and targets a critical epigenetic regulator, Ezh2, which methylates histone H3. Our preliminary data show that Ezh2 expression is inversely regulated with miR101 and is critical for normal skeletal development, based on our phenotypic characterization of a conditional Ezh2 null mouse model. We will determine (i) the biological significance of miR155 in a novel miR/TF circuit that suppresses osteoblast differentiation in cell culture and in transgenic mouse models that conditionally express miR155 (Aim 1), as well as (ii) the importance of a miR101-Ezh2 axis during osteoblast differentiation in vivo and ex vivo by conditional expression of miR101 in transgenic mice (Aim 2). Upon completion of the primary objectives of this proposal, my long term goals are to examine the molecular consequences of miRNA dependent TF-EpiReg networks using RNASeq and ChiP-Seq approaches to define new drug-sensitive regulatory pathways. These studies should ultimately lead to the development of osteotropic drugs that could become new bone anabolic therapies. This future work as independent investigator would leverage my combined expertise in pharmacology, epigenetics and mouse bone biology that I acquired during doctoral and post-doctoral training.

Public Health Relevance

Osteoporosis, osteoarthritis and tendonitis are debilitating diseases that affect millions of Americans with billions of dollars spent annually on osteoporotic fractures. Mesenchymal progenitor cell are precursor to musculoskeletal lineage cells including osteoblasts, chondrocytes and tenocytes and myoblasts. The expression of key transcription factors is critical in determining the fate of mesenchymal progenitor cells. Transcription factors and other genes are epigenetically regulated by microRNAs. We will evaluate the role of select microRNAs during osteoblast development. Understanding the interplay between transcription factors and microRNAs is instrumental in deciphering the mechanism that control bone development and diseases. Transcription factors and microRNAs may be targeted in the future in the treatment of osteoporosis and other musculoskeletal diseases.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F10B-B (20)L)
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Chen, Faye H
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Mayo Clinic, Rochester
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United States
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Samsonraj, Rebekah M; Dudakovic, Amel; Manzar, Bushra et al. (2018) Osteogenic Stimulation of Human Adipose-Derived Mesenchymal Stem Cells Using a Fungal Metabolite That Suppresses the Polycomb Group Protein EZH2. Stem Cells Transl Med 7:197-209
Camilleri, Emily T; Dudakovic, Amel; Riester, Scott M et al. (2018) Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affect cartilage development. J Biol Chem 293:19001-19011
Sterner, Rosalie M; Kremer, Kimberly N; Dudakovic, Amel et al. (2018) Tissue-Nonspecific Alkaline Phosphatase Is Required for MC3T3 Osteoblast-Mediated Protection of Acute Myeloid Leukemia Cells from Apoptosis. J Immunol 201:1086-1096
Soreide, Endre; Denbeigh, Janet M; Lewallen, Eric A et al. (2018) Fibrin glue mediated delivery of bone anabolic reagents to enhance healing of tendon to bone. J Cell Biochem 119:5715-5724
Dudakovic, Amel; Camilleri, Emily T; Paradise, Christopher R et al. (2018) Enhancer of zeste homolog 2 (Ezh2) controls bone formation and cell cycle progression during osteogenesis in mice. J Biol Chem 293:12894-12907
Pollock, Kathryn; Samsonraj, Rebekah M; Dudakovic, Amel et al. (2017) Improved Post-Thaw Function and Epigenetic Changes in Mesenchymal Stromal Cells Cryopreserved Using Multicomponent Osmolyte Solutions. Stem Cells Dev 26:828-842
Riester, Scott M; Torres-Mora, Jorge; Dudakovic, Amel et al. (2017) Hypoxia-related microRNA-210 is a diagnostic marker for discriminating osteoblastoma and osteosarcoma. J Orthop Res 35:1137-1146
Riester, Scott M; Denbeigh, Janet M; Lin, Yang et al. (2017) Safety Studies for Use of Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells in a Rabbit Model for Osteoarthritis to Support a Phase I Clinical Trial. Stem Cells Transl Med 6:910-922
Dudakovic, Amel; Gluscevic, Martina; Paradise, Christopher R et al. (2017) Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing. Gene 609:28-37
Galeano-Garces, Catalina; Camilleri, Emily T; Riester, Scott M et al. (2017) Molecular Validation of Chondrogenic Differentiation and Hypoxia Responsiveness of Platelet-Lysate Expanded Adipose Tissue-Derived Human Mesenchymal Stromal Cells. Cartilage 8:283-299

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