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Molecular Alterations in Bone Development and Bone

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Molecular Alterations in Bone Development and Bone MOLECULAR ALTERATIONS IN BONE DEVELOPMENT AND BONE TUMORIGENESIS DISSERTATION Presented in Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Emilia Mahoney, M.D. Molecular, Cellular and Developmental Biology Graduate Program The Ohio State University 2009 Dissertation Committee: Lawrence Kirschner, M.D., PhD, Advisor Rebecca Jackson, M.D. Michael Ostrowski, PhD Thomas Rosol, D.V.M., PhD Copyright by Emilia Mahoney 2009 ABSTRACT Bone is a specialized connective tissue that forms, together with the cartilage, the skeletal system. These tissues play important roles in locomotion, protection of vital organs and in metabolism. The fundamental constituents in bone are the extracellular matrix and the cells. The extracellular matrix is formed by collagen fibers and noncollagenous proteins. The bone cells are represented by osteoblasts, osteocytes and osteoclasts. There are many genetic alterations that can affect either the developing bone leading to skeletal deformities or the mature bone, leading to a vast array of pathological entities among which the tumors represent a significant percentage. The work presented here is focused on both these aspects by studying Gremlin regulation during limb development and Prkar1a mutation that causes formation of unique bone tumors through PKA dysregulation. Chapter 2 comprises a study on the developmental failure of distal limb structures seen in the mouse limb deformity ( ld ) phenotype. This anomaly is caused by the loss of Gremlin in the limb buds either through mutation of Grem1 or by loss of a transcriptional global control region (GCR) located in the neighboring Fmn1 gene. This study describes a new allele of ld due to complete deletion of Fmn1 , including its GCR, thus providing information about the role of this transcriptional global control region in Gremlin expression as well as long-range transcriptional effects that extend beyond Fmn1 and Grem1. ii Chapter 3 includes a histological characterization of the bone tumors that develop in Carney complex patients and initial studies describing the molecular alterations associated with the tumor phenotype. Carney complex (CNC) is an autosomal dominant familial multiple neoplasia syndrome that may be caused by inactivating mutations in PRKAR1A , the gene encoding the type 1A regulatory subunit of protein kinase A (PKA). Loss of this regulatory subunit causes increased PKA activity in CNC tumors. In this study, I used Prkar1a +/- mice, generated previously in our lab as a model for the human tumor syndrome CNC. Bone lesions in these mice are closely analogous to those observed in human patients and the lesions arise from incompletely differentiated cells of the osteoblast lineage. The observations of this study led to the conclusion that tumor formation in this system is driven mainly by excess PKA activity, along with other growth factors, such as FGF, IL6 and members of the Wnt pathway. In chapter 4, I examined the role of Wnt signaling in these bone tumors induced by the PKA dysregulation. Previous studies showed that PKA is able to phosphorylate beta-catenin at two sites, which appeared to be unrelated to the beta-catenin ubiquitination. In the bone tumor cells, beta-catenin forms nuclear speckles that co- localize with PML protein (promyelocytic leukemia protein). The speckled pattern and the co-localization of beta-catenin with PML protein seem to be induced by the increased PKA activity, as suggested by the studies performed on tumor osteoblasts and on wild- type osteoblasts treated with Forskolin, a PKA inducer. Further analysis will be necessary to define the precise role and the consequences of the beta-catenin – PML co-localization. In addition, the experimental data in this study suggests a novel, PKA-dependent, iii transcriptional activity of beta-catenin at the Cebpd, Cbfa2 and Ezrin promoter regions, and of the PML protein at Wnt5a promoter region. Collectively, the results of the first study bring a deeper understanding of the process of limb development. The second and third studies establish the CNC bone tumors as the first in vitro system aimed at directly studying the biochemical alterations associated with tumorigenesis caused by PRKAR1A mutations. The investigation of the bone tumors provides insights not only in osteoblast biology but also in the general question of tumorigenesis associated with the CNC. iv Dedicated to my family and friends v AKNOWLEDGEMENTS First, I would like to thank my advisor, Dr. Lawrence Kirschner. A physician and a researcher in the same time, you showed me how the clinical thinking can combine with science to start from interesting ideas and to follow with valuable research data. From the very beginning you encouraged me to come up with my own ideas and also to learn how to create the appropriate plan that would take me on the journey of experiments toward the rewarding destination of good data. Despite your busy schedule, you have been available to answer my innumerable questions and to offer guidance whenever was necessary. I would also like to thank past and present members of the Kirschner lab: Will, Kiran, Georgette and Zhirong for making the work atmosphere enjoyable, for sharing ideas and for the fun times outside the lab. Special thanks to Drs Rebecca Jackson, Michael Ostrowski and Thomas Rosol for making time in their busy schedule to serve on my committee and for their helpful advice. To all my friends who made easier the transition from life in Romania to Columbus, US: thank you. Nesrine, Nael, Jan, Karla, Gina, Catalina, Francisco, Cheryl, Mary – your friendship means a lot to me. vi And finally, I can not thank enough my parents Ion and Mariana and my husband Jeff for believing in me, for their constant love and support and for always being proud of me. vii VITA October 10, 1974...........................................................Born in Bucharest, Romania September 1989 – June 1993 ........................................Victor Babes Nursing High School Bucharest, Romania September 1993 – June 1999 ........................................Medical School Independent University “Titu Maiorescu” Bucharest, Romania September 1999 – December 2000...............................Internship “Coltea” General Hospital Bucharest, Romania January 2001 – September 2003...................................Internal Medicine Residency “Floreasca” Emergency Hospital Bucharest, Romania September 2003 – Present.............................................PhD Candidate, MCDB Program, The Ohio State University viii PUBLICATIONS Pavel E, Zhao W, Powell KA, Weinstein M, Kirschner LS. Analysis of a new allele of limb deformity (ld) reveals tissue- and age-specific transcriptional effects of the Ld Global Control Region. Int J Dev Biol. 2007;51(4):273-81. Pavel E, Nadella K, Towns WH 2nd, Kirschner LS. Mutation of Prkar1a causes osteoblast neoplasia driven by dysregulation of protein kinase A. Mol Endocrinol. 2008 Feb;22(2):430-40. Nadella K, Saji M, Jacob NK, Pavel E, Ringel MD , Kirschner LS. Regulation of actin dynamics and migration by Protein Kinase A-mediated phosphorylation of Limk1. Accepted for publication in EMBO Reports March 2009. Kirschner LS, Yin Z, Jones G, Mahoney E. Mouse Models of Altered Protein Kinase A Signaling. Accepted for publication in Endocrine Related Cancer April 2009. FIELD OF STUDY Major Field: Molecular, Cellular and Developmental Biology ix TABLE OF CONTENTS ABSTRACT........................................................................................................................ ii ACKNOWLEDGEMENTS............................................................................................... vi VITA................................................................................................................................ viii LIST OF FIGURES .......................................................................................................... xii LIST OF ABBREVIATIONS........................................................................................... xv CHAPTER 1 ........................................................................................................................1 INTRODUCTION ...............................................................................................................1 1.1 Bone.............................................................................................................1 Bone as an organ macroscopic organization................................................1 Bone as a tissue – microscopic organization...............................................2 Bone formation............................................................................................3 1.2 Limb development.......................................................................................5 Formation of limb buds................................................................................5 Formation of skeletal elements....................................................................9 Roles of Formin and Gremlin in limb development are revealed by analysis of murine limb deformity (ld) malformations ................................9 1.3 Osteochondromyxomas – bone tumors that develop in Carney complex patients.......................................................................................................13 Clinical aspects of Carney Complex..........................................................13 Bone tumors in Carney Complex
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