Our proposed studies will investigate the central hypothesis that epigenetic enzymes regulate lineage- commitment and maturation of bone forming cells in vivo. Our studies will inform new strategies for bone anabolic therapies aimed at reducing fracture risk. Current therapies have major clinical side-effects and patient restrictions. Yet, with the aging demographics of the US population, there is a significant urgency to develop new strategies that promote bone accrual. In addition, there are a range of short term anabolic applications (e.g., spine fusion, radiation induced bone loss) that would benefit from new mechanism-based approaches. The proposed work will provide (i) proof of concept for leveraging epigenetic drugs to promote bone accrual in a pre-translational setting, but also (ii) fundamental insights into the role of chromatin heterochromatinization in controlling bone formation.
Aim 1 will assess the in vivo bone stimulatory effects of conditional ablation of the gene for histone methyl transferase EZH2 during post-natal development and in skeletally mature mice in which the EZH2 gene is deleted in mesenchymal stem cells or osteoblasts. These studies will reveal whether genetic loss of EZH2 stimulates bone accrual, is osteoprotective (after estrogen-depletion in ovariectomized mice), and/or accelerates bone healing. These genetic studies will follow-up on our preliminary data indicating that pharmacological inhibitors with established safety profiles (e.g., EZH2 inhibitor GSK126) can be used to promote bone accrual in vivo and osteoblast differentiation in vitro.
Aim 2 will examine the molecular basis for the bone stimulatory effects of EZH2 inactivation. Using genomics approaches (ChIP-seq and RNA-seq), we will define and functionally test signaling molecules and gene regulatory factors that respond to EZH2 inhibition. We will also specifically investigate the attractive model that EZH2 inhibition initiates a sustained bone anabolic response by enhancing endogenous paracrine signaling of osteogenic ligands (e.g., BMP, WNT and PTH). We will focus specifically on examining the molecular basis for our new observation that GSK126 and BMP2 synergize in osteoblast maturation. Our findings have important clinical ramifications by generating genetic support for considering epigenetic drugs in promoting new bone formation. Conceptual innovation of these studies will be obtained by molecular analysis of the epigenetic regulatory role for EZH2 which permits definition of early mechanistic events that control osteogenic differentiation and osteoblast maturation.

Public Health Relevance

The US population us advancing with age and healthy elderly adults maintain more active life styles, while fracture risk increases with age due to gradual bone degeneration. Current therapies that seek to reduce bone loss or improve bone mineral density can have major clinical side-effects or cause patient discomfort, and they cannot be used on all patients. Therefore, there is a significant need to find new methods for improving bone quality that are based on a thorough understanding of how bone forms. This project has identified a bone stimulatory drug that is already safely used in the clinic for other indications. This drug (GSK126) targets an enzyme (Enhancer of Zeste Homolog-2, EZH2) that controls the molecular memory (epigenetics) of osteoblasts by generating tiny molecular bookmarks (lysine methylation) on proteins that control packaging of DNA into either readable (?open chromatin?) or unreadable (?heterochromatin?) regions. At present, our mechanistic understanding of EZH2 is limited and does not yet permit consideration of clinical applications. However, our findings to date are encouraging and indicate that inhibition of EZH2 function is remarkably osteogenic and stimulates bone accrual, while blocking this protein does not affect other musculoskeletal lineages (e.g., cartilage and tendon). We are now using EZH2 inhibition as a tool to understand at what developmental stages it acts to regulate bone formation (Aim 1) and precisely through which molecular networks the enzyme achieves its biological effects (Aim 2). By modulating the activity of this enzyme through multiple loss of function approaches, we will be able to establish which main signaling pathways are controlled by EZH2 as a molecular blueprint for the conversion of immature mesenchymal cells to osteoblasts that support formation of bone. EZH2 is the first epigenetic regulator known to suppress osteoblastogenesis, and therefore its inhibition can activate bone formation through ?double-negative? stimulation. Hence, this proposal characterizes a ?first-in- class? druggable epigenetic enzyme and as such is expected to have a major impact on clinical strategies to control fracture risk and a range of short-term bone anabolic applications. Furthermore, these studies will address the fundamentally important biological question as to how humans grow and maintain bone tissue through novel epigenetic regulatory pathways.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR049069-14A1
Application #
9387067
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Chen, Faye H
Project Start
2003-09-30
Project End
2022-08-31
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
14
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
Paradise, Christopher R; Galeano-Garces, Catalina; Galeano-Garces, Daniela et al. (2018) Molecular characterization of physis tissue by RNA sequencing. Gene 668:87-96
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
Salib, Christopher G; Lewallen, Eric A; Paradise, Christopher R et al. (2018) Molecular pathology of adverse local tissue reaction caused by metal-on-metal implants defined by RNA-seq. Genomics :
Riester, Scott M; Lin, Yang; Wang, Wei et al. (2018) RNA sequencing identifies gene regulatory networks controlling extracellular matrix synthesis in intervertebral disk tissues. J Orthop Res 36:1356-1369
Araya, H├ęctor F; Sepulveda, Hugo; Lizama, Carlos O et al. (2018) Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2. J Cell Biochem 119:8204-8219
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
Morrey, Mark E; Sanchez-Sotelo, Joaquin; Lewallen, Eric A et al. (2018) Intra-articular injection of a substance P inhibitor affects gene expression in a joint contracture model. J Cell Biochem 119:1326-1336
Lewallen, Eric A; Salib, Christopher G; Trousdale, William H et al. (2018) Molecular pathology of total knee arthroplasty instability defined by RNA-seq. Genomics 110:247-256
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

Showing the most recent 10 out of 171 publications