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Estrogen receptor-related receptor gamma (ERR) is a regulator of skeletogenesis by Marco Cardelli A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Medical Biophysics University of Toronto © Copyright by Marco Cardelli 2015 Estrogen receptor-related receptor gamma (ERR) is a regulator of skeletogenesis Marco Cardelli Doctor of Philosophy Medical Biophysics University of Toronto 2015 Abstract Sex steroids, such as Estrogen, play important roles in physiological and pathological processes, including bone growth and bone homeostasis. Estrogen's effects are mediated through its receptors, Estrogen receptor alpha and beta (ER and ER). Despite its importance, loss of expression of ERs does not result in severe skeletal effects, suggesting that other factors are involved in the Estrogen pathway(s). Estrogen receptor related receptors (ERRs) are a family of orphan nuclear receptors, i.e., with no known natural ligands, comprising ERRα, ERRβ, and ERRγ. While ERR has been shown to be involved in various aspects of skeletogenesis, there is very little know about the potential role of ERR. To address this question, I used gain-of- function and loss-of-function approaches in mice. In the gain-of-function model with cartilage- specific ERR-overexpression, the size of the craniofacial, axial and appendicular skeletons was reduced compared to that in wild type (WT) mice. Histological analysis revealed a reduction in the length of the transgenic versus WT growth plate, attributable to a reduced proliferative zone accompanied by a decrease in the number of Ki67-positive proliferating cells, with no significant change in apoptosis. Quantification of expression of putative target genes suggested that ERRγ negatively regulates chondrocyte proliferation and positively regulates matrix synthesis to ii coordinate growth plate height and organization. In an ERRγ global knockout mouse line, I observed no gross morphological anomalies or difference in skeletal length in newborn mice, but by 8 weeks of age ERRγ +/- male but not female mice exhibited increased trabecular bone, which was further increased by 14 weeks. Histomorphometric and serum biochemistry data indicated that the trabecular bone increase was due to an increase in bone formation, with no apparent change in bone resorption. Analysis of ERRγ +/- versus ERRγ +/+ bone marrow stromal cell cultures indicated that ERRγ negatively regulates osteoblast differentiation and matrix mineralization but not mesenchymal precursor number. These data, together with results from co-immunoprecipitation and Runx2 antisense oligonucleotide treatment experiments in vitro, indicated that ERRγ is not required for skeletal development but is a sex-dependent negative regulator of postnatal bone formation, acting in a RUNX2- and apparently differentiation stage-dependent manner. I conclude that ERRγ plays negative regulatory roles in both cartilage and bone in postnatal mice and may be an important new therapeutic target in skeletal diseases and conditions. iii Acknowledgments I would like to thank my supervisor, Dr. Jane Aubin. Dr. Aubin is not only an expert in the fields of bone and cartilage biology as well as mesenchymal stem cells, she is an inspirational mentor and leader. It was a great privilege to have had the opportunity to study in the Aubin lab. I would like to thank the members of the Aubin lab: Tanya Zappitelli, Ralph A. Zirngbl, Ruolin Guo, Frieda Chen, Jonathan F. Boetto and Kristen P. McKenzie for constructive discussion and critique. Special gratitude to Tanya for her many hours of counsel and support, and to Ruolin for her assistance in experimental procedures. Very many thanks to my committee members, Dr. Armen Manoukian and Dr. Eldad Zacksenhaus for their guidance and support throughout my PhD training. iv Table of Contents Acknowledgments .......................................................................................................................... iv Table of Contents ............................................................................................................................ v List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................. x Chapter 1 ......................................................................................................................................... 1 1 Introduction ................................................................................................................................ 1 1.1 Bone Development .............................................................................................................. 1 1.1.1 Endochondral Ossification ...................................................................................... 1 1.1.2 Intramembranous Ossification ................................................................................ 4 1.2 Chondrogenesis/chondrocyte differentiation ...................................................................... 7 1.2.1 Growth factors ........................................................................................................ 7 1.2.2 Cyclins .................................................................................................................. 10 1.2.3 Extracellular matrix proteins ................................................................................. 10 1.2.4 Transcription factors ............................................................................................. 11 1.3 Osteogenesis/Osteoblast Differentiation ........................................................................... 13 1.3.1 Growth factors ...................................................................................................... 13 1.3.2 Transcription factors ............................................................................................. 16 1.4 Estrogen in skeletal development ..................................................................................... 17 1.4.1 Effect of Estrogen and its receptors ...................................................................... 17 1.4.1.1 Longitudinal growth ............................................................................... 17 1.4.1.2 Bone remodeling .................................................................................... 19 1.5 Estrogen receptor-related receptors .................................................................................. 23 1.5.1 ERR .................................................................................................................... 27 1.5.1.1 ERR and cartilage ................................................................................ 27 v 1.5.1.2 ERR and bone ...................................................................................... 28 1.5.2 ERR ..................................................................................................................... 29 1.5.3 ERR ..................................................................................................................... 30 1.5.3.1 ERRand energy metabolism ................................................................ 30 1.5.3.2 ERRand skeletogenesis ........................................................................ 31 1.6 Objectives ......................................................................................................................... 34 Chapter 2 ....................................................................................................................................... 35 2 Cartilage-specific overexpression of ERR results in chondrodysplasia and reduced chondrocyte proliferation ......................................................................................................... 35 2.1 Abstract ............................................................................................................................. 36 2.2 Introduction ....................................................................................................................... 37 2.3 Material and Methods ....................................................................................................... 40 2.3.1 Ethics Statement .................................................................................................... 40 2.3.2 Construction of pCol2a1mERRγ2 and generation of transgenic mice ................. 40 2.3.3 LacZ stain for detection of transgene .................................................................... 41 2.3.4 Gene expression analysis ...................................................................................... 41 2.3.5 Western Blotting ................................................................................................... 41 2.3.6 Whole mount skeletal staining .............................................................................. 42 2.3.7 Histological and immunofluorescence analysis .................................................... 42 2.3.8 TUNEL assay ........................................................................................................ 44 2.3.9 Expression analysis of putative target genes ........................................................ 44 2.3.10 Statistical Analysis ...............................................................................................
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