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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32AR066508-03
Application #
9271083
Study Section
Special Emphasis Panel (ZRG1-F10B-B (20)L)
Program Officer
Chen, Faye H
Project Start
2015-05-20
Project End
2018-05-19
Budget Start
2017-05-20
Budget End
2018-05-19
Support Year
3
Fiscal Year
2017
Total Cost
$65,250
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
Other Domestic Non-Profits
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
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
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
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
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
Khani, Farzaneh; Thaler, Roman; Paradise, Christopher R et al. (2017) Histone H4 Methyltransferase Suv420h2 Maintains Fidelity of Osteoblast Differentiation. J Cell Biochem 118:1262-1272
Morhayim, Jess; van de Peppel, Jeroen; Dudakovic, Amel et al. (2017) Molecular characterization of human osteoblast-derived extracellular vesicle mRNA using next-generation sequencing. Biochim Biophys Acta 1864:1133-1141
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
Camilleri, Emily T; Gustafson, Michael P; Dudakovic, Amel et al. (2016) Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production. Stem Cell Res Ther 7:107
Dudakovic, Amel; Camilleri, Emily T; Riester, Scott M et al. (2016) Enhancer of Zeste Homolog 2 Inhibition Stimulates Bone Formation and Mitigates Bone Loss Caused by Ovariectomy in Skeletally Mature Mice. J Biol Chem 291:24594-24606
Morhayim, Jess; van de Peppel, Jeroen; Braakman, Eric et al. (2016) Osteoblasts secrete miRNA-containing extracellular vesicles that enhance expansion of human umbilical cord blood cells. Sci Rep 6:32034

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