Expression of Capn6 During Osteoclast Differentiation in Wild-type and HDAC4 Knockout Mice A THESIS SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Molly Kopf IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF DENTISTRY Kim Mansky, PhD; Amy Tasca, DDS, PhD; and John Beyer, DDS, PhD June 2019 © Molly Kopf 2019 Acknowledgements First, I would like to thank my thesis committee chair Dr. Kim Mansky for her time and dedication to this project. It would not have been successful without her support and guidance. I would also like to thank my other committee members Dr. Amy Tasca and Dr. John Beyer for all their help and input for my master’s thesis. I greatly appreciate the time they committed to reviewing and shaping this thesis. Additionally, I would like to express my gratitude to those individuals working in the Mansky lab at the University of Minnesota. They taught me several lab procedures, contributed to the study by providing samples, and created a fun yet productive environment for completing my research. These individuals include but are not limited to Dr. Eric Jensen, Bora Faulkner, Kristina Astleford, Andrew Norton and Emily Campbell. Furthermore, I’d like to thank the full-time and part-time faculty members of the orthodontic clinic for their commitment to my education over the last two years. I’m eager to use the wisdom and knowledge I gleaned from them in my future career. Lastly, I’d like to acknowledge my fellow orthodontic co-residents. I value their support and will always cherish the memorable experience of our orthodontic residency. i Dedication I dedicate my master’s thesis to my husband Blake. Even though we were separated by hundreds of miles, you were there supporting me through it all. ii Abstract Introduction: The process of osteoclast differentiation and function consists of a network of complex signaling pathways with multiple negative and positive regulators. Previous studies suggest histone deacetylase (HDAC) proteins play a suppressive role in osteoclast differentiation; however, not much is known about the specific role of HDAC4. Expression of calpain 6 (Capn6) has been linked to increased organization of osteoclast microtubules for bone resorption. In this study, we observe the expression of Capn6 in wild-type and HDAC4 knockout osteoclasts. Methods: qRT-PCR was preformed to assess Capn6 expression in wild-type and HDAC4 knockout mice over days 0 to 4 of osteoclast differentiation. Immunoblot analysis was used to assess CAPN6 levels in both groups. Results: Levels of Capn6 expression increased later in osteoclast differentiation in the wild-type osteoclasts, though the results were not significant. There was a significant increase in Capn6 in osteoclasts from HDAC4 knockout mice after 3 days of RANKL stimulation. This was also significant when comparing HDAC4 knockout to wild-type osteoclasts. Conclusion: HDAC4 may be a negative regulator of osteoclast function, suppressing the expression of Capn6. More studies are indicated to understand the interaction of HDAC4 and Capn6 in the regulation of osteoclast activity. iii Table of Contents List of Tables v List of Figures vi Introduction 1 Materials and Methods 12 Results 15 Discussion 21 Conclusions 24 References 25 iv List of Tables Table 1. Capn6:Hprt expression in ten biologically independent wild-type 17 mice samples Table 2. Capn6:Hprt expression in five biologically independent HDAC 4 19 knockout mice samples v List of Figures Figure 1. Stimulation of osteoclast precursor by RANKL and M-CSF 3 Figure 2. The signaling pathways involved in the activation of NFATc1 5 Figure 3. Process of osteoclast resorption with HCl acid and 7 Cathepsin K Figure 4. Expression of RNA sequencing genes in wild-type vs. knockout 16 mice on day 2 of osteoclast differentiation Figure 5. A) Immunoblot analysis of Capn6 in wild-type mice B) Mean 18 Capn6:Hprt expression in wild-type mice over four days of differentiation Figure 6. Mean Capn6:Hprt expression over days of differentiation 19 between wild-type and HDAC4 knockout mice Figure 7. Mean Capn6:Hprt expression over days of differentiation 20 between wild-type (black) and HDAC4 knockout mice (white) vi Introduction Bone is often thought of as the strong, rigid supporting structure of the bodies of various organisms. It is necessary for the vitality of those organisms allowing for mobility, protection of significant organs, production of blood cells, and storage of minerals like calcium. Bone tissue is made up of a collagenous matrix, inorganic elements, and cells. Even though bone is strong and rigid, it is not static. Throughout a lifetime, bone is constantly being reshaped, remodeled, and repaired. This continual dynamic process of resorption (bone degradation) and apposition (bone formation) is completed at such a rate that in one year, approximately 10% of the bone content is replaced in a human adult.1 Bone remodeling cycle The process of resorption and apposition is facilitated by specific cells called osteoclasts and osteoblasts, respectively. Through various signaling pathways, bone remolding occurs first by osteoclasts recruitment to the site of repair. Resorption creates Howship’s lacunae on the bony surface. Once these lacunae get to a depth of about 50µm, certain signals recruit mesenchymal cells to differentiate into osteoblasts at the site and start bone apposition.2 A disruption in the process of removing and adding bone can cause harmful effects in humans.2,3 For example, an increase in osteoclastic activity will lead to an imbalance where more bone will be resorbed. This will weaken the bone and cause bone disorders such as osteoporosis, rheumatoid arthritis, periodontal disease, multiple myeloma, and certain metastatic cancers.1–5 A decrease in 1 resorption processes by osteoclasts leads to an increase in the amount of bone and cause diseases such as osteopetrosis.5,6 Osteoclast differentiation Since osteoclasts play a key role in most bone disorders, studies have been conducted to learn more about their origin and differentiation process.4,5 Osteoclasts are multinuclear giant cells derived from hematopoietic precursors of the monocyte and macrophage lineage that surround the bone.3 Original studies looked at failed osteoclast recruitment leading to osteopetrosis to determine transcription factors involved in differentiation. One of the first transcription factors that plays a role in osteoclastogenesis is PU.1.4,5,7 This is a positive regulator of transcription involved in the generation of the common progenitors for both osteoclasts and macrophages; therefore, a deletion of PU.1 will cause a lack of both of those cell types, and knockout mice exhibit an osteopetrotic phenotype.1,7 In addition, many genes required for osteoclast and macrophage differentiation have PU.1 binding sites in their promoters.7 Studies have shown that there are two main factors that stimulate osteoclastogensis in vitro: macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL)1–3. RANKL is a membrane-bound cytokine derived from the tumor necrosis factor (TNF) family.1– 3 Osteoprotegerin (OPG), another TNF-derived receptor, was used to discover the importance of RANKL in osteoclast differentiation.4 OPG was simultaneously discovered by two groups: Amgen and Snow Brand Milk Group.6 It is a secreted 2 protein and was found to be a decoy receptor competing with RANK to bind RANKL, and thus inhibiting osteoclast differentiation and inducing osteopetrosis in vitro and in vivo. 2,5,8 Both RANK and RANKL are found in other organs of the body such as skeletal muscle, thymus, liver, colon, adrenal glands, lungs, brain and kidneys.6 The OPG/RANK/RANKL regulation system is important in the regulation of resorption by negatively and positively controlling the activation of RANK on osteoclasts. Figure 12: Stimulation of osteoclast precursor by RANKL and M-CSF PU.1 stimulates expression of colony-stimulating factor 1 receptor (CSF1R) on macrophage progenitors. This is a receptor for CSF1, also known as M-CSF which causes proliferation of osteoclast precursor cells as well as upregulates RANK. Again, M-CSF knockout mice have a lack of proliferating osteoclast cells, leading to an increase in bone density.7 M-CSF also activates microphthalmia-associated transcription factor (MITF), which plays a key role in 3 later stages of osteoclastogensis. MITF regulates the expression of anti-apoptotic protein Bcl-2 and promotes the survival of osteoclasts and macrophages.1,5,7 RANKL leads to osteoclast differentiation through its complex signaling pathway9. Both osteoblasts and osteocytes have been shown to be the source of RANKL that binds to RANK in osteoclasts.10,11 In vivo, it was demonstrated that a direct contact with osteoblasts or osteocytes is not needed for RANKL to bind to their receptors on the osteoblast progenitor cells.3,12 Additionally, it has been discovered that vesicular RANK secreted by osteoclasts exhibits a reverse signaling pathway that in turn upregulates osteoblast differentiation.12 This shows the unique coupling of bone formation and bone apposition. Therefore, therapies that inhibit osteoclast differentiation will also decrease the amount of bone formation due to this coupling phenomenon. Once RANK and RANKL bind, RANK has the ability to bind TRAF6 in osteoclasts, which has a major role in signal transduction for osteoclast differentiation.1,2,13 TRAF6 activates NF-κB and mitogen-activated kinases (MAPKs) pathway which led to activations