The goals of this study are to determine the functional roles of the epigenetic de novo DNA methyltransferase enzyme, Dnmt3b, in regulating post-natal articular cartilage homeostasis. From this work, we expect to identify new targets for the design of novel strategies to treat osteoarthritis (OA), the most costly condition in the US Medicare population. The pathogenesis of OA is still poorly understood and there is an unmet clinical need to discover new therapies to slow down or stop the cartilage degradation that occurs in this disease. Epigenetic changes (e.g. DNA methylation, histone modifications, microRNA-mediated regulation) are implicated in many diseases, including OA. In fact, recent genome wide methylation profiling has revealed differentially methylated loci in cells of healthy and OA cartilage. Of the two de novo DNA methyltransferase enzymes, we found that Dnmt3b protein, but not Dnmt3a, was localized in chondrocytes of healthy murine and human articular cartilage. Importantly, we also showed that Dnmt3b expression decreased in aging/OA cartilage and that transgenic mice with Dnmt3b loss-of-function in chondrocytes developed spontaneous OA. Thorough analysis of RNA-Seq and Methyl-Seq data generated from normal versus Dnmt3b knock-down chondrocytes revealed that a potential downstream target of Dnmt3b is the metabolic enzyme, 4 aminobutyrate aminotransferase (Abat). Abat functions to metabolize gamma-aminobutyric acid (GABA) into succinate and is a key regulator of mitochondrial metabolism in the cell. Interestingly, we found that an FDA-approved Abat inhibitor drug, vigabatrin, was able to attenuate catabolic gene expression that was induced in Dnmt3b loss-of- function chondrocytes in vitro. Altogether, our preliminary data suggest the existence of a Dnmt3b/Abat axis in chondrocytes and that modulation of this axis may be a promising therapeutic strategy to treat OA. In vitro and in vivo approaches will be utilized to modulate Dnmt3b or Abat expression and/or function to define mechanisms involved in their regulation of articular chondrocytes and their role in the development OA. Two main Specific Aims are proposed.
Specific Aim 1 will involve in vivo post-natal ablation of Dnmt3b in chondrocytes to determine if mice develop spontaneous OA during aging or following joint destabilization induced by meniscal ligament injury. In vitro experiments will be performed to show that Abat is a critical downstream target of Dnmt3b in the regulation of cell metabolism and in the differentiation of articular chondrocytes into a hypertrophic/catabolic phenotype.
Specific Aim 2 will utilize Dnmt3b gain-of-function models in vitro and in vivo to determine if Dnmt3b over-expression confers protection against OA. Vigabatrin will be administered to determine if in vivo inhibition of Abat can delay the onset of OA in mice following joint injury. In summary, this program will define Dnmt3b-mediated epigenetic changes, Abat function and cell metabolism as a novel pathway axis in the development of OA. This work will enhance our understanding of mechanisms regulating OA and provide novel targets for innovative therapeutic approaches to treat OA.

Public Health Relevance

Diseases of cartilage affect growth and development and, in the elderly, results in the development of osteoarthritis. The treatment of osteoarthritis is currently the most costly single medical condition in the elderly population in the United States. The main goals of this study are to determine the role of the de novo DNA methyltransferase enzyme, Dnmt3b, in regulating post-natal articular cartilage homeostasis. The focus of the grant is to define the epigenetic regulation of articular chondrocytes by reduced Dnmt3b expression as a key mechanism involved in the development of osteoarthritis. Completion of these studies will identify Dnmt3b, and the factors it regulates, as innovative targets for the prevention and treatment of osteoarthritis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR069605-03
Application #
9416924
Study Section
Skeletal Biology Structure and Regeneration Study Section (SBSR)
Program Officer
Lester, Gayle E
Project Start
2016-03-01
Project End
2021-02-28
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Washington University
Department
Orthopedics
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Shen, Jie; Abu-Amer, Yousef; O'Keefe, Regis J et al. (2017) Inflammation and epigenetic regulation in osteoarthritis. Connect Tissue Res 58:49-63
Shen, Jie; Wang, Cuicui; Li, Daofeng et al. (2017) DNA methyltransferase 3b regulates articular cartilage homeostasis by altering metabolism. JCI Insight 2:
Wang, Cuicui; Abu-Amer, Yousef; O'Keefe, Regis J et al. (2017) Loss of Dnmt3b in Chondrocytes Leads to Delayed Endochondral Ossification and Fracture Repair. J Bone Miner Res :