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The Role of Peroxisome in Osteoblast Differentiation and Functions

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The role of peroxisomes in osteoblast differentiation and functions Inaugural Dissertation submitted to the Faculty of Medicine in partial fulfillment of the requirements for the PhD-Degree of the Faculties of Veterinary Medicine and Medicine of the Justus Liebig University Giessen By Fan, Wei Of Zhengzhou China Giessen 2014 1 From the Institute for Anatomy and Cell Biology, Division of Medical Cell Biology Director/Chairperson: Prof. Dr. Eveline Baumgart-Vogt of the Faculty of Medicine of Justus Liebig University Giessen First Supervisor and Committee Member: Prof. Dr. Eveline Baumgart-Vogt Second Supervisor and Committee Member: Prof. Dr. Martin Diener Examination chair and Committee Member: Prof. Dr. Klaus-Dieter Schlüter Date of Doctoral Defense: 22nd September 2015 2 Declaration “I declare that I have completed this dissertation single-handedly without the unauthorized help of a second party and only with the assistance acknowledged therein. I have appropriately acknowledged and referenced all text passages that are derived literally from or are based on the content of published or unpublished work of others, and all information that relates to verbal communications. I have abided by the principles of good scientific conduct laid down in the charter of the Justus Liebig University of Giessen in carrying out the investigations described in the dissertation.” Giessen, November 25th 2014 Fan, Wei 3 Table of Contents 1. Literature Review and Introduction .............................................................................5 1.1. The peroxisome structure, functions and biogenesis ...........................................5 1.1.1. Basic morphology and metabolic function of peroxisomes ......................................... 5 1.1.2. Peroxisome biogenesis ..................................................................................................... 8 1.1.3. Enzyme composition and metabolic functions in peroxisomes ................................ 19 1.2. Peroxisome biogenesis disorders .......................................................................... 28 1.2.1. The peroxisome biogenesis disorders and human diseases .................................... 28 1.2.2. Animal models for peroxisome biogenesis disorders ................................................. 29 1.3. Cells of the bone and their role in bone remodeling and the pathogenesis of osteoporosis .................................................................................................................... 32 1.3.1 The major types of bone cells and functions ................................................................ 32 1.3.2. Osteobiogenesis, osteoblast differentiation and functional alterations ................... 34 1.4. Bone phenotype and peroxisomal phenotype in the Sirt1 KO mouse model ... 38 2. Aims of the study ......................................................................................................... 40 3. Materials and Methods ................................................................................................ 42 3.1. General Materials used in the laboratory .............................................................. 42 3.1.1. Chemicals for the general application of molecular and morphological experiments ....................................................................................................................................................... 42 3.1.2. Laboratory general instruments ..................................................................................... 44 3.1.3. The General materials for cell culture .......................................................................... 46 3.2. Experimental animals ............................................................................................... 47 3.2.1. Pex11β KO and Pex13 KO animals in Germany ........................................................ 47 3.2.2 Sirt1 whole body KO mice experiment in the USA ...................................................... 47 3.3. Methods ..................................................................................................................... 48 3.3.1. Primary osteoblasts and MC3T3-E1 pre-osteoblast-like fibroblast cell line ........... 48 3.3.2. Transfection of MC3T3-E1 cells and primary osteoblast ........................................... 50 3.4. Molecular biological experiments........................................................................... 52 3.4.1. RNA isolation from primary osteoblast and MC3T3-E1 cell culture and the reverse transcription to generate cDNA ................................................................................................ 52 1 3.4.2. Semi-quantitative polymerase chain reaction and agarose gel electrophoresis. .. 52 3.4.3. Quantitative real time-polymerase chain reaction ...................................................... 55 3.4.4. Osteoblast protein abundance analysis via Western blots ....................................... 56 3.5 Morphological experiments ...................................................................................... 61 3.5.1. Indirect immunofluorescence stainings of primary osteoblasts ................................ 61 3.5.2. Indirect immunofluorescence staining on Paraformaldehyde-fixed paraffin- embedded mouse tissue ........................................................................................................... 62 3.5.3. Apoptosis detection by terminal deoxynucleotidyl transferase dUTP nick end labeling ......................................................................................................................................... 63 3.5.4. Whole skeleton bone volume and bone density estimation using flat-panel volumetric computed tomography (fpvCT) of P0.5 mouse pups. ........................................ 64 3.6. Functional assay of protein DNA binding, biophysical methodology ............... 65 3.6.1. Dual-Luciferase reporter gene assay to monitor transcriptional activities of markers of bone differentiation RUNX2 and FoxOs, PPARs. .............................................. 65 3.6.2. Biophysical Methodology to determine cell cycle of primary osteoblasts ............... 67 3.6.3. Flow cytometric analysis of ROS in Sirt1 gene shRNA knockdown MC3T3-E1 cells ....................................................................................................................................................... 69 3.7 Numerical data presentation and statistical analysis ........................................... 70 4. Results: ......................................................................................................................... 71 4.1. Part I: The Pex11β gene KO causes peroxisomal dysfunction, resulting in osteoblast differentiation delay and severe cell signaling alterations. .................... 71 4.1.1. Pex11β KO in primary osteoblasts and Pex11β gene knockdown via Pex11β shRNA in the MC3T3-E1 cell line ............................................................................................. 71 4.1.2. The Pex11β gene KO results in dramatically decreasing the expression level of the peroxisomal functional enzymes........................................................................................ 73 4.1.3. Osteoblast differentiation and maturation were severely altered in the primary osteoblast cells culture with Pex11β KO ................................................................................. 76 4.1.4. The DNA binding functions of key nuclear receptors regulating the osteoblast differentiation and function were significantly altered in Pex11β KO osteoblasts. ........... 78 4.1.5. Significantly higher amount of apoptosis of primary Pex11β KO compared to WT osteoblasts. .................................................................................................................................. 82 4.1.6. Strong disruption of the cell cycle and proliferation rate in Pex11β KO osteoblast 84 2 4.2. Part II: The Pex13 KO caused peroxisomal biogenesis defect result in the interference with the antioxidative response, osteoblast differentiation and maturation ......................................................................................................................... 86 4.2.1. Pex13 KO mice (P0.5) exhibit a delay in the ossification process ........................... 86 4.2.2. Peroxisomal biogenesis genes and the osteoblast differentiation process were disturbed. ..................................................................................................................................... 91 4.2.3. The oxidative response is stimulated by oxidative stress in the bone cells of Pex13 KO mice due to peroxisomal dysfunction ................................................................................ 92 4.2.4. The DNA binding activity of the Runx2 nuclear receptor which regulates osteoblast differentiation and function was significantly attenuated in primary osteoblast of Pex13 KO mice........................................................................................................................................ 95 4.3. Part III: SIRT1 deficiency impairs peroxisomal functions and promotes pre- mature differentiation of pre-osteoblasts and abnormal bone development in mice ............................................................................................................................................ 97 4.3.1. SIRT1 protein level is altered in primary osteoblast with peroxisomal deficiency.
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