Research Collection Doctoral Thesis Differentially load-regulated gene expression in mouse trabecular osteocytes Author(s): Wasserman, Elad Publication Date: 2010 Permanent Link: https://doi.org/10.3929/ethz-a-006128911 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH No. 18938 Differentially Load-Regulated Gene Expression in Mouse Trabecular Osteocytes A dissertation submitted to ETH Zurich for the degree of Doctor of Sciences presented by ELAD WASSERMAN M.Sc. Cell Biology and Histology, Tel-Aviv University born 27th September, 1972 citizen of Israel accepted on the recommendation of Examiner: Prof. Dr. Ralph Müller Co-Examiner: Prof. Dr. Itai Bab 2010 Table of contents: Acknowledgements………………………………………………………………………………….v Summary……………………………………………………………………………………………vi Zusammenfassung………………………………………………………………………………….ix 1. Introduction……………………………………………………………………………………...13 1.1. Hypotheses and specific aims……………………………………………………………….15 1.1.1. Developing method of isolating mRNA from trabecular osteocytes…………………..16 1.1.2. Identify load-induced differentially regulated genes…………………………………..16 1.2. Outline of the thesis…………………………………………………………………………17 2. Background……………………………………………………………………………………...21 2.1. Bone anatomy……………………………………………………………………………….22 2.2. Osteocytes as mechanosensors/endocrine and paracrine organ …………………………….26 2.3. Bone remodeling…………………………………………………………………………….33 2.4. Mechanical load-induced bone adaptation .…………………………………..…………….40 2.5. Mouse genetics……………………………………………………………………………...45 3. Developing a method for isolation of osteocyte RNA………………………………………....67 3.1. Separation of trabecular bone from caudal vertebra………………………………………...68 3.2. Enzymatic digestion of non-osteocytic cells………………………………………………..70 3.3. RNA extraction from denuded trabeculae…………………………………………………..71 3.4. Comparative marker gene mRNA expression in enzymatically isolated cell fractions and extracted RNA from denuded trabecular bone…………………………………………75 4. Load-induced differential regulation mRNA of trabecular osteocytes……………………...89 4.1. Single loading………………………………………………………………………………89 4.2. Repetitive loading…………………………………………………………………………..99 4.3. Functional genomics for identification of load-regulated pathways……………………...107 4.4. Confirmation of individual load-regulated genes in single loading………………………116 5. Synthesis………………………………………………………………………………………..127 Appendix…………………………………………………………………………………………..133 A1-A4. List of up- and down-regulated genes………………………………………………...134 A5-A6. List of load-regulated signalling pathways………………………………………........184 Curriculum Vitae ………………………………………………………………………………...190 Acknowledgements The work presented in this thesis is the direct result of a great team effort and I am gratefully indebted to every member of that team. First of all I would like to express my deep gratitude to both my supervisors, Professor Dr. Ralph Müller and Professor Dr. Itai Bab. Without their passion, guidance and leadership the successful realization of this project would not have been possible. Furthermore, I would like to thank them for their time, and the open door policy for which I am eternally grateful. I would also like to thank PD Dr. Franz Weber (University of Zürich) and Dr. Haike Hall-Bozic for their support and for providing me facilities in their laboratories. Special thanks go to Dr. Duncan Webster for all of his help in doing the many experiments. His company and expertise were invaluable and significantly contributed to the outcome of this thesis. I also would like to thank Dr. Gisela Kuhn and Floor Lambers for their help during loading studies. During my thesis, I enjoyed a fruitful close collaboration with The Functional Genomics Center Zurich (FGCZ) located at the University of Zurich. Many members of this institute made it possible to perform differential expression microarrays and bioinformatics statistical analyses. Particular thanks go to the past and present members of the Institute of Biomechanics. Their expertise and companionship created a pleasant environment in which to work. I sincerely hope to stay in contact and share some good times with you in the future. I am deeply grateful to my parents. Their unconditional love and support have enabled me to achieve all my goals. These few words cannot even begin to describe my gratitude and appreciation for all they have done for me. Finally, the financial support of the Swiss National Science Foundation (SNF) and the Swiss Federal Institute of Technology (ETHZ) is gratefully acknowledged. - v- Summary In light of the many bone diseases and injuries for which proper treatment is yet to be developed, there is a significant need to further address new approaches to stimulate bone healing. Osteoporosis is a disease characterized by an excessive decrease in bone mass which can lead to an increased susceptibility to fractures, skeletal deformation and, in more severe cases, death owing to morbidity. The disease has been attributed to both genetic and age-related factors. There are various medications available, which have been shown to delay bone loss, however no cure is yet achievable. To treat the disease, medical research is attempting to target genes which define osteoporosis, using the mouse as a model. Owing to the recent deciphering of the mouse genome and the high homology that exists between human and mouse genomes, inbred strains of mice represent ideal models for genetic studies. Using the mouse to identify genes implicated in the bone remodeling process could lead to advances in understanding that enable the precise regulation of genes and proteins responsible for particular bone phenotypes, i.e. bone mineral density or bone strength. One interesting phenotype under investigation is the response of bone to mechanical loading or its “mechano-sensitivity”. Mechanical loading is perhaps the most important single physiological/environmental factor regulating bone mass and shape. Age-related bone loss and consequent osteoporosis have been attributed, at least in part, to a reduction in muscle mass/function and the resultant decrease in mechanical usage of the skeleton. On the other hand, mechanical overloading has been shown to enhance bone formation and cause a net gain in cancellous bone mass, the major structural component of skeletal load-bearing sites. However, very little is known about the mechanisms involved in the load-induced anabolic effects in trabecular bone, mainly due to the lack of in vivo models to study load-induced molecular events. Building on the studies investigating the effect of mechanical loading on trabecular bone adaptation in the C56BL/6 mouse tail model that was recently developed in our group, the next long-term objective of this thesis was to elucidate the molecular mechanisms involved in the osteogenic anabolic effect of mechanical loading and to find genes and gene pathways that are regulated by mechanical loading. Mice have a well-characterized genome accessible to manipulations by transgenic and knockout technologies. An understanding of the molecular pathways governing load-stimulated bone formation could provide opportunities to mimic or augment bone mechano-sensitivity using pharmacological and molecular agents thereby leading to the development of novel strategies in the management of osteoporosis and other skeletal deficits. - vi- To investigate the genetic regulation of mechanical loading, an ex-vivo method was established for the isolation of representative samples of mouse vertebral intact ribonucleic acid (RNA) derived selectively from trabecular osteoblast/lining cells and osteocytes, by using sequential collagenase digestions and pulverization. High quality total RNA preparations were isolated immediately following cell separation using conventional reagents and protocols. The quantity of total RNA isolated from trabecular osteocytes of a single caudal vertebra was sufficient for further differential gene expression analysis. To investigate a single and repetitive mechanical load-induced differential gene expression, the fifth caudal vertebra (C5) of C57BL/6 (B6) female mice was mechanically stimulated by respective single or repetitive load doses, each dose consisting of 3’000 cycles at a frequency of 10 Hz with an amplitude of 0N and 8N via two pins inserted into the adjacent vertebrae (C4 and C6). Mice were sacrificed six hours after the last mechanical loading and high quality total RNA preparations were analyzed for gene expression arrays, using Affymethrix Mouse Genome chips. Differential gene expression analysis of a single mechanical load revealed a total of 331 significantly regulated genes (P < 0.05), including 281 up-regulated probes and 50 down-regulated probes. Also, functional genomics analysis of acute loading, using GeneGo MetaCore software, indicated 65 load-regulated molecular pathways in which significantly regulated probes were present. In particular, up- regulation of insulin growth factor 1 (IGF-1, 2.2 fold) and wingless-type MMTV integration site family, member 5a (Wnt5a, 3.4 fold) genes have been shown which are thought to be activators of osteoblasts differentiation. In contrast, down-regulation of WNT inhibitor factor 1 gene (WIF-1, 1.8 fold) was shown, an inhibitor of WNT/beta-cathenin pathway for activation of osteoblast differentiation. Differential gene expression analysis of repetitive mechanical loading (three times per week