The Regulation and Function of BMPs in Osteoclastogenesis A Dissertation SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Raphael Edward Huntley IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Raj Gopalakrishnan DMD, PhD, Adviser Kim Mansky PhD, Co Adviser June 2019 Raphael Huntley 2019 ã Acknowledgements Thank you to my committee members, Dr. Rajaram Gopalakrishnan, Dr. Kim Mansky, Dr. Paul Jardine, and Dr. Michael O’Connor. Without your input, guidance, and dedication this thesis would not be possible. Thank you to Dr. Mark Herzberg and Ann Hagen, whose efforts have created and sustained the MinnCReST training program, which provided me financial and developmental support as well as career guidance and training. i Dedication To my parents, who tried their best to raise me like a mensch. It almost worked. To my loving wife, who may be the only person to read it cover to cover. To my siblings, who have always referred to me as ‘genius boy, Rafi’. To my dog; he really is a good boy. ii Table of Contents: Acknowledgements .......................................................................................................................... i Dedication .......................................................................................................................................... ii Table of Contents: .......................................................................................................................... iii Table to Figures: ............................................................................................................................. vi Chapter 1 ............................................................................................................................................ 1 Introduction, Statement of Purpose, and Specific Aims ................................................... 1 1.1 Introduction ............................................................................................................................... 2 1.1.1 Clinical use and clinical complications of BMPs ......................................................... 3 1.1.2 Osteoclast Biology............................................................................................................... 3 1.1.3 BMP Structure and Function ............................................................................................ 6 1.1.4 BMP Ligands expression in osteoclasts ........................................................................ 8 1.1.4.1 BMP1 ................................................................................................................................................................ 8 1.1.4.2 BMP2 ................................................................................................................................................................ 8 1.1.4.3 BMP3 ................................................................................................................................ 10 1.1.4.4 BMP4 .............................................................................................................................................................. 10 1.1.4.5 BMP5 .............................................................................................................................................................. 11 1.1.4.6 BMP6 .............................................................................................................................................................. 11 1.1.4.7 BMP7 .............................................................................................................................................................. 12 1.1.4.8 BMP8 .............................................................................................................................................................. 12 1.1.4.9 BMP9 .............................................................................................................................................................. 12 1.1.5 BMP Inhibitors .................................................................................................................. 13 1.1.6 BMP Receptors .................................................................................................................. 14 1.1.6.1 BMPR2 ........................................................................................................................................................... 14 1.1.6.2 BMPR1A ........................................................................................................................................................ 14 1.1.6.3 BMPR1B ........................................................................................................................................................ 15 1.1.7 BMP Signaling ................................................................................................................... 15 iii 1.1.7.1 Noncanonical Signaling ............................................................................................................................ 15 1.1.7.2 Canonical Signaling ................................................................................................................................... 16 1.1.8 Conclusions ........................................................................................................................ 18 1.2 Statement of Purpose ......................................................................................................... 18 1.2.1 Significance of Research .............................................................................................................................. 18 1.2. Hypothesis ............................................................................................................................ 19 1.3 Specific Aims ......................................................................................................................... 19 1.3.1 Determine the functional significance of glycosylated amino acids in Twisted Gastrulation regulation of osteoclast differentiation ............................................................................................................ 19 1.3.2 Determine if Twisted gastrulation null osteoclasts are hypersensitive to M-CSF and RANKL signaling ...................................................................................................................................................................... 20 1.3.3 Characterize the skeletal phenotype of a mouse model null for Bmp2 in myeloid cells ....... 20 Chapter 2 ......................................................................................................................................... 22 The Function of Twisted Gastrulation in Regulating Osteoclast Differentiation is Dependent on BMP Binding ...................................................................................................... 22 2.1 Introduction .......................................................................................................................... 23 2.2 Material and Methods ........................................................................................................ 25 2.2.1 Mice .................................................................................................................................................................... 25 2.2.2 Harvesting of Bone Marrow and Generation of Osteoclast Cells .................................................. 25 2.2.3 Osteoclast Transduction ............................................................................................................................. 26 2.2.4 RNA Isolation and Quantitative RT-PCR ................................................................................................ 26 2.2.5 Immunoblotting ............................................................................................................................................. 27 2.2.6 TRAP Stain ....................................................................................................................................................... 28 2.2.7 Quantitating Nuclei ....................................................................................................................................... 28 2.2.8 Resorption Assays ......................................................................................................................................... 28 2.2.9 Statistics ........................................................................................................................................................... 29 2.3 Results .................................................................................................................................... 29 2.3.1 Engineering and Expression of TWSG1 constructs in OCLs............................................................ 29 2.3.2 Loss of TWSG1 binding to BMPs results in larger OCLs ................................................................... 30 2.3.3 TWSG1 mutants have increased resorptive activity ......................................................................... 32 2.3.4 Effect of W66 and N80Q/N146Q on OCLs is mediated through modulation of BMP signaling ...................................................................................................................................................................... 33 2.3.5 TWSG1