Dissertations / Theses on the topic 'Bone resorption – Molecular aspects'

[Truncated abstract] Osteoclasts are multinucleated giant cells responsible for the resorption of the mineralized bone matrix during the process of bone remodelling. During activation towards bone resorption, polarization of the osteoclast results in the formation of a unique plasma membrane, the ruffled border, the actual resorptive organelle of the osteoclast. Through this domain protons are actively pumped into the resorption lacuna creating an acidic microenvironment that favours the dissolution of the mineralized bone matrix. The polarised secretion of protons is carried out by the action of the vacuolar-type (H+)-ATPase (V-ATPase), composed of functionally and structurally distinct subunits of the V1 and V0 domains. The general structure of the V-ATPase complex is highly conserved from yeast to mammals, however, multiple isoforms for specific V-ATPase subunits do exist exhibiting differential subcellular, cellular and tissue-specific localizations. This study focuses on the molecular identification and characterization of V-ATPase accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit in osteoclasts. Using the techniques of cDNA Subtractive Hybridization and DNA Micro-Array analyses respectively, the accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit were identified in RAW264.7-cells derived OcLs. ... Using web-based computational predictions, two possible transmembrane domains, an N-terminus 'signal anchor' sequence and a C-terminus dilysine- like endoplasmic reticulum (ER) retention signal were identified. By confocal microscopy, EYFP-tagged e was found to localize to the perinuclear region of transfected COS-7 cells in compartments representing the ER and Golgi apparatus with some localization in late endosomal/lysosomal-like vesicles. ER truncation of e did not alter its subcellular localization but exhibited significantly weaker association with Ac45 compared to the wild-type as depicted by BRET analyses. Association with the other V0 subunits remain unaffected. This may hint at a possibility that Ac45 may play a role in the masking of the ER signal of e following it's incorporation into the V0 domain. Although no solid evidence for a role in the assembly of the mammalian VATPase have been established, subunit e still represents a potential candidate whose role in the V-ATPase complex requires further investigation. Collectively, the data presented in this thesis has provided further insight into the composition of the osteoclast V-ATPase proton pump by: 1) identifying an accessory subunit, Ac45 which shows promise as a potential candidate for the regulation and/or targeting of the V-ATPase complex in osteoclasts and truncation of its targeting signal impairs osteoclastic bone resorption; 2) identification and preliminary characterization of the d2 isoform of the V0 domain d subunit whose exact role in the V-ATPase complex and in osteoclasts remains to be determined, although its has been implicated to be essential for osteoclastic function; and 3) Preliminary characterization of subunit-e, a potential assembly factor candidate for the mammalian V-ATPase V0 domain.

[Truncated abstract] Bone remodeling is intricately regulated by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The elevation in osteoclast number and/or activity is a major hallmark of several common pathological bone disorders including post-menopausal osteoporosis, osteoarthritis, Paget's disease, and tumour-mediated osteolysis. Receptor activator of nuclear factor kappa B ligand (RANKL) is a key cytokine for osteoclast differentiation and activation. The association of RANKL to its cognate receptor, RANK, which is expressed on osteoclast precursors and mature osteoclasts, is essential for osteoclast formation and activation. The intimate interaction between RANKL and RANK triggers the activation of a cascade of signal transduction pathways including NF-κB, NFAT, MAPK and PI3 kinase. Although osteoclast signaling pathways have been intensively studied, the precise molecules and signaling events which underlie osteoclast differentiation and function remain unclear. In order to dissect the molecular mechanism(s) regulating osteoclast differentiation and activity, this thesis herein explores the key RANKL/RANK-mediated signaling pathways. Four truncation mutants within the TNF-like domain of RANKL [(aa160-302), (aa160-268), (aa239-318) and (aa246-318)] were generated to investigate their potential binding to RANK and the activation to RANK-signal transduction pathways. All were found to differentially impair osteoclastogenesis and bone resorption as compared to the wild-type RANKL. The impaired function of the truncation mutants of RANKL on osteoclast formation and function correlates with their reduced ability to activate crucial RANK signaling including NF-κB, IκBα, ERK and JNK. Further analysis revealed that the truncation mutants of RANKL exhibited differentially affinity to RANK as observed by in vitro pull-down assays. ... It is possible that Bryostatin 1 acts via upregulation of a fusion mechanism as the RANKL-induced OCLs are morphologically enlarged, exhibiting increased nuclei number expressing high level of DC-Stamp. Furthermore, Rottlerin was shown to inhibit NF-κB activity, whereas Bryostatin 1 showed the opposing effects. Both inhibitor and activator were also found to modulate other key osteoclastic signaling pathways including NFAT and total c-SRC. These findings implicate, for the first time, Protein Kinase C delta signaling pathways in the modulation of RANKL-induced osteoclast differentiation and activity. Taken together, the studies presented in this thesis provide compelling molecular, biochemical and morphological evidence to suggest that: (1) RANKL mutants might potentially serve as peptide mimic to inhibit RANKL-induced signaling, osteoclastogenesis and bone resorption. (2) A cross talk mechanism between extracellular Ca2+ and RANKL exist to regulate on osteoclast survival. (3) TPA suppressed RANKL-induced osteoclastogenesis predominantly during the early stage of osteoclast differentiation via modulation of NF-κB. (4) Selective inhibition of Protein Kinase C signaling pathways involved in osteoclastogenesis might be a potential treatment method for osteoclast-related bone diseases. (5) Protein Kinase C delta signaling pathways play a key role in regulating osteoclast formation and function.

Osteoclasts are multinucleated cells found exclusively in bone and are derived from the haematopoietic cells of monocytes/macrophage lineage. The cell-to-cell interaction between osteoblastic/stromal cells and osteoclast precursor cells is necessary for osteoclastogenesis. Receptor Activator of NF-κB ligand (RANKL) was identified as a membrane-bound TNF ligand family member that is the ‘master’ cytokine expressed on osteoblastic/stromal cells, which stimulate osteoclastogenesis through cell-to-cell contact with osteoclast precursors. RANKL is considered to be a factor that is necessary and sufficient for the induction of osteoclastogenesis (Lacey, et al., 1998). RANKL is a type II transmembrane cytokine of the TNF ligand superfamily and has an active TNF-like core domain at the extracellular domain. This active TNF-like core domain is thought to be the region through which it binds to it’s active receptor, RANK, for the activation of signal transduction pathways for the initiation of processes leading to osteoclastogenesis (Lacey, et al., 1998; Li, et al., 1999). It was hypothesized that any change in the active TNF-like core domain might affect the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. Hence, this thesis sought to investigate the effects of changes in the active TNF-like core domain by truncation mutation on the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. A cDNA fragment encoding the full-length TNF-like core domain of rat RANKL (rRANKL) (aa160-318) was cloned into the bacterial expression pGEX vectors and stably expressed in Eschechia coli as a fusion protein with the C-terminus of glutathione S-transferase (GST). Four mutants (aa160-302, aa160-268, aa239-318 and aa246-318) were also generated by truncation mutation in the TNF-like core domain, and cloned into the pGEX vector to produce GST-rRANKL mutants. The proteins were over-expressed and affinity purified to 95% in purity. GST-rRANKL (160-318) containing the full length TNF-like core domain was able to induced osteoclastogenesis in spleen cells in the presence of M-CSF and in RAW264.7 cells in the absence of M-CSF. It was also found to activate mature osteoclast activity in vitro, ex vivo and in vivo. It has the highest binding affinity to RANK and the greatest potency for NF-κB activation as well as the induction of osteoclastogenesis compared to the truncated mutants. Mutants generated by truncation of the TNF-like core domain revealed that the TNF-like core domain is important for the interaction with the RANK, for high binding affinity, NF-κB activation and induction of osteoclastogenesis. In general, the truncated mutants not only displayed a reduction in the binding affinity to RANK, but also a reduction in NF-κB activation, and significantly reduced potency in the induction of osteoclastogenesis. Interestingly, mutant GST-rRANKL (160-268) showed a higher affectivity than the other mutants did, in that it had greater binding affinity to RANK, and in NF-κB activation than the rest of the truncated mutants. Mutants GST-rRANKL (239-318) and GST-rRANKL (246-318) on the other hand, showed little potency in the induction of osteoclast formation, however, might have an inhibitory effect through competition with full length GST-rRANKL (160-318) as well as inducing a response in vivo resulting in an increase in the serum calcium level. In conclusion, this thesis demonstrated that the TNF-like core domain of RANKL is active, and imperative in the binding to RANK, activating signal transduction pathways and induction of osteoclastogenesis. Changes in the active TNF-like core domain affected the ability, affinity and efficiency of RANKL binding to the receptor, RANK and consequently affected the activation of signal transduction pathways and osteoclastogenesis.

In cases of compromised bone remodeling like osteoporosis, insufficient osseointegration occurs and results in implant failure. Implant retention relies on proper secondary fixation, which is developed during bone remodeling. This process is disrupted in metastatic bone diseases like osteoporosis. Osteoporosis is characterized low bone mass and bone strength resulting from either accelerated osteoclast-mediated bone resorption or impaired osteoblast-mediated bone formation. These two processes are not independent phenomena. In fact, osteoporosis can be viewed as a breakdown of the cellular communication connecting bone resorption to bone formation. Because bone remodeling occurs at temporally generated specific anatomical sites and at different times, local regulators that control cross-talk among the cells of the BRU are important. Previous studies show Ti implant surface characteristics like roughness, hydrophilicity, and chemistry influence the osteoblastic differentiation of human MSCs and maturation of OBs. Furthermore, microstructured Ti surfaces modulate the production of factors shown to be important in the reciprocal communication necessary for the maintenance of healthy bone remodeling. Semaphorin signaling proteins are known to couple the communication of osteoblasts to osteoclasts and are capable of stimulating bone formation or bone resorption depending on certain cues. Implant surface properties can be optimized to exploit these effects to favor rapid osseointegration in patients with osteoporosis.

Age-related bone loss is a serious public health problem affecting 15 to 20 million people in the United States (1). In the last decade, several studies have investigated the effects of repeated bouts of exercise on bone density. Although this increase in bone density is mediated via hormones and/or cellular metabolites few studies have linked the increase in bone density with changes in these hormones or metabolites. Therefore, the purpose of this study was to determine the effect of a single bout of weight bearing exercise on the growth factors that alter bone metabolism. Seven healthy female subjects (age 23 ± 5 yrs.) agreed to participate in this study. A V02 max test was administered to each subject to determine their overall fitness level. A treadmill speed corresponding to 70% of their V02 max was then developed and the subjects ran for 40 minm on the treadmill at that speed. Blood samples were drawn pre and 1, 2, 3, 4, 5, 10, 15, 20, 30, 45, and 60 min post-exercise and assayed for serum growth hormone, calcium, parathyroid hormone, and alkaline phosphatase. No significant change was found in parathyroid hormone, calcium, or alkaline phosphatase serum levels. Growth hormone was found to be significantly decreased 30, 45, and 60 min. post-exercise as compared to pre-exercise levels (7.089, 5.399, 4.728, and 11.28 ng/mL, respectively). While this indicates that exercise may cause a depression in growth hormone levels during recovery, the elevated pre-exercise levels of three of the seven subjects masked the exercise stimulated release of growth hormone. Growth hormone stimulates bone absorption via insulin-like growth factors and the GH data suggests that these hormones may have been released well after our last measurement time point. In conchusion, 40 minutes of running exercise at 70% of V02 max does not produce a change in any of the hormones or metabolic factors directly associated with calcium turnover in the bone, suggesting that acute exercise has no immediate effect on bone metabolism. However, the exercise protocol did stimulate GH release which influences bone growth indirectly by its ability to regulate IGF release. Therefore, even though no acute effects were evident, a single bout of exercise may alter the long term control of bone metabolism.<br>School of Physical Education

Indiana University-Purdue University Indianapolis (IUPUI)<br>Signal Transducer and Activator of Transcription 3 (STAT3) is known to be related to bone metabolism. Mutation of STAT3 causes a rare disorder in which serum levels of IgE are elevated. This causes various skeletal problems similar to osteoporosis. To examine the effect of STAT3 in the osteoclast, we obtained two osteoclast specific STAT3 knockout mouse models: one using the CTSK promoter to drive Cre recombinase and another using a TRAP promoter. Examination of these mice at 8 weeks of age revealed a decreased trabecular bone volume in CTSK specific STAT3 knockout mice along with a slight decrease in osteoclast number in both CTSK and TRAP specific STAT3 knockout females. We also noticed changes in bone mineral density and bone mechanical strength in females. These data suggest that STAT3 plays a part in the function of the osteoclast.

Indiana University-Purdue University Indianapolis (IUPUI)<br>Heritable disorders of phosphate handling are the most common cause of hypophosphatemic rickets in developed countries. Isolated renal phosphate wasting and subsequent low serum phosphate concentrations may result from a number of genetic disorders that include: autosomal dominant hypophosphatemic rickets (ADHR), X-linked hypophosphatemic rickets (XLH), and autosomal recessive hypophosphatemic rickets (ARHR). Fibroblast growth factor-23 (FGF23), identified as the causative gene in ADHR, is produced in bone and plays a central role in kidney phosphate regulation. Increased serum concentrations of FGF23 lead to renal phosphate wasting through down regulation of renal sodium-phosphate co-transporters. However, the molecular mechanisms of FGF23 bioactivity in hormonal phosphate regulation are largely unknown. An experimental focus of this dissertation was to investigate the molecular mechanisms of FGF23-mediated phosphate regulation in the bone-kidney hormonal axis. To this end, the role of Dentin Matrix Protein 1 (DMP1), newly identified as the gene responsible for ARHR, was further defined by the identification of a novel large deletion as well as testing the molecular consequences of DMP1 mutations. FGF23 requires a signaling complex composed of Klotho and an FGFR for bioactivity, however, the location and composition of the signaling complex is unknown. Klotho localizes to the renal distal convoluted tubule, whereas the sodium phosphate co-transporters are expressed within the renal proximal tubules. The molecular mechanisms of FGF23 signaling were investigated by isolating a novel marker of FGF23 bioactivity using array technology, determining the location of initial FGF23 signaling in the kidney, and by identifying a novel mutation in a receptor upstream of FGF23 production. Taken together, these results increase the knowledge of the molecular mechanisms of phosphate homeostasis in relation to FGF23 bioactivity, leading to the identification of potentially novel therapeutic targets.<br>indefinitely

背景：纤维化是各种因素导致肾脏慢性损伤的最终病理过程，是决定肾功能转归的关键因素。肌纤维母细胞作为构成肾脏纤维化组织的主要细胞成分，其来源尚不清楚。本研究认为骨髓来源的巨噬细胞向肌纤维母细胞转分化（MMT）可能是肾脏纤维化中肌纤维母细胞的主要来源。我们分别在慢性肾脏病患者的肾活检组织和小鼠单侧输料管梗阻模型（UUO）中验证这一假说。<br>方法：我们用激光共聚焦技术和流式细胞染色的方法检测小鼠UUO肾脏和患者肾活检组织中的MMT细胞(F4/80⁺α-SMA⁺或CD68⁺α-SMA⁺)。为了验证骨髓来源的MMT在肾纤维化中的重要作用，UUO模型分别在以下小鼠进行：1）去除骨髓的C57BL/6J小鼠，给予或不给予绿色荧光蛋白（GFP）标记的骨髓细胞移植；2）GFP⁺骨髓的嵌合体小鼠；3）巨噬细胞敲除或不敲除的lysM-Cre/DTR小鼠；4）GFP⁺Smad3⁺/⁺ 或GFP⁺Smad3⁻/⁻骨髓的嵌合体小鼠。我们用实时定量PCR和Western blot检测小鼠肾组织collagen-I和α-SMA水平。另外，我们观察MMT细胞和PDGFR-β⁺ pericytes, CD45⁺collagen I⁺ fibrocytes的关系。最后，通过观察GFP⁺Smad3⁻/⁻骨髓嵌合体小鼠UUO模型肾纤维化程度和TGF-β1刺激下TGF-β受体II或Smad3敲除的骨髓巨噬细胞MMT的不同进一步探索TGF-β/Smad3通路对MMT的影响。<br>结果：去除骨髓后，肾脏collagen-I沉积和α-SMA⁺肌纤维母细胞生成显著受抑制，骨髓细胞移植可以恢复肾脏纤维化，免疫荧光染色显示嵌合体小鼠中多数（80-90%）肌纤维母细胞来自于骨髓巨噬细胞转分化。同时，在白喉霉素诱导的巨噬细胞敲除小鼠中，50-60%巨噬细胞被去除，伴有纤维化明显减少，并且和MMT细胞显著减少相关。进一步验证巨噬细胞通过MMT直接参与肾脏纤维化过程。患者肾活检组织亦可见不同数目MMT细胞，纤维化活跃组织中MMT细胞可占到肌纤维母细胞总数的80%。另外，我们发现无论在小鼠模型还是患者肾活检组织中，多数MMT细胞表达pericyte（PDGFR-β⁺）和fibrocyte（CD45⁺collagen-I⁺）标记物。Smad3⁻/⁻骨髓嵌合体小鼠肾纤维化程度明显低于Smad3⁺/⁺骨髓嵌合体组，TGF-β1刺激下TGF-β受体II或Smad3敲除的骨髓巨噬细胞MMT明显低于不敲除组，提示TGF-β/Smad3通路在MMT过程中起重要作用。<br>结论：骨髓来源的MMT是肾纤维化组织中肌纤维母细胞的主要来源，TGF-β/Smad3 通路在MMT 过程中起重要作用。<br>Background: Fibrosis is the ultimate pathological feature and determinant process for chronic kidney disease (CKD) regardless of the underlying etiology. Myofibroblasts are a key cell type in renal fibrosis by producing excessive collagen matrix. However, the origin of myofibroblasts during renal fibrosis remains largely controversial. This thesis tested the hypothesis that bone marrow (BM)-derived macrophage myofibroblast transition (MMT) may be a key pathway leading to renal fibrosis in patients with CKD and in a mouse model of unilateral ureteral obstructive nephropathy (UUO).<br>Methods: Renal fibrosis was assessed by expression of fibrotic marker collagen I and α-SMA using real-time PCR and western-blot analysis. MMT was determined in both mouse and human kidneys by confocal microscopy and flow cytometry with α-SMA⁺F4/80⁺ (or CD68⁺). The critical role of BM-derived MMT in renal fibrosis was examined in a mouse model of UUO, with various conditions: 1) BM depletion followed by BM transplantation (BMT) with GFP⁺ BM cells; 2) in GFP⁺ BM chimeric mice; 3) in lysM-Cre/DTR mice with or without inducible macrophage deletion; 4) in GFP⁺Smad3⁺/⁺ or GFP⁺Smad3⁻/⁻ BM chimeric mice. In addition, MMT was also validated in renal biopsy tissues from patients with different forms of CKD. Further more, we also studied the relationship between MMT and PDGFR-β⁺ pericytes or CD45⁺collagen I⁺ fibrocytes in both human and mouse fibrotic kidneys. Finally, mechanisms of MMT was examined in the UUO kidney induced in GFP⁺Smad3⁻/⁻ BM chimeric mice and in BM macrophages lacking TGF-β receptor II or Smad3.<br>Results: As described in Chapter III, mice with BM deletion were protected from renal fibrosis as demonstrated by blocking α-SMA⁺ myofibroblasts and collagen I accumulation. In contrast, BMT restored renal fibrosis in UUO kidney, demonstrating the critical role for BM cells in renal fibrosis. Importantly, the majority (85-90%) of α-SMA⁺ myofibroblasts were derived from BM macrophages as identified by GFP⁺F4/80⁺α-SMA⁺ revealing BM-macrophages given rise to myofibroblasts via MMT during kidney fibrosis. Similarly, MMT appeared as a major pathway of myofibroblast origin in patients with CKD, accounting for up to 80% of total myofibroblasts in the active stage of tissue fibrosis and fibrocellular crescents. To test the function role of macrophages in renal fibrosis via MMT, macrophages were conditionally deleted from the UUO kidneys in lysM-Cre/DTR mice as shown in Chapter IV, deletion (50-60%) of macrophages resulted in inhibition of MMT and renal fibrosis. Unexpectedly, most MMT cells (80-90%) were shown to co-express the pericyte marker (PDGFR-β⁺) and fibrocyte markers (CD45⁺collagen I⁺) in both human CKD and UUO (Chapter V), suggesting a BM macrophage origin for pericytes and fibrocytes during renal fibrosis. Finally, TGF-β/Smad3 appeared to be a mechanism driven MMT because mice and BM macrophages lacking either Smad3 or TβRII were protected against MMT and progressive renal fibrosis in the UUO kidney and in vitro.<br>Conclusions: MMT is derived from BM macrophages and regulated by TGF-β/Smad3. MMT is a major pathway of myofibroblast origin during renal fibrosis in both human and animal model of CKD.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Wang, Shuang.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 161-179).<br>Abstracts also in Chinese.<br>Chapter ABSTRACT --- p.ii<br>Chapter DECLARATION --- p.viii<br>Chapter ACKNOWLEDGEMENTS --- p.ix<br>Chapter TABLE OF CONTENTS --- p.xi<br>Chapter LIST OF ABBREVIATION --- p.xv<br>Chapter LIST OF FIGURES AND TABLES --- p.xvii<br>Chapter CHAPTER I --- p.1<br>INTRODUCTION --- p.1<br>Chapter 1. 1 --- Renal fibrosis and myofibroblasts --- p.2<br>Chapter 1. 1. 1 --- Pathology of renal fibrosis --- p.2<br>Chapter 1. 1. 2 --- The generation and modulation of myofibroblasts. --- p.3<br>Chapter 1. 1. 2. 1 --- EMT and EndMT --- p.5<br>Chapter 1. 1. 2. 2 --- Pericytes --- p.8<br>Chapter 1. 1. 2. 3 --- Fibrocytes --- p.16<br>Chapter 1. 2 --- Role of macrophage in fibrogenesis --- p.21<br>Chapter 1. 3 --- TGF-β signaling pathway in renal fibrosis --- p.23<br>Chapter 1. 3. 1 --- TGF-β superfamily --- p.23<br>Chapter 1. 3. 2 --- TGF-β/Smad signaling pathway --- p.24<br>Chapter CHAPTER II --- p.29<br>MATERIALS AND METHODS --- p.29<br>Chapter 2. 1 --- Materials --- p.30<br>Chapter 2. 1. 1 --- Regents and equipments --- p.30<br>Chapter 2. 1. 1. 1 --- Regents and equipment for mouse genotyping --- p.30<br>Chapter 2. 1. 1. 2 --- Regents and equipments for real-time PCR --- p.30<br>Chapter 2. 1. 1. 3 --- Reagents and equipments for immunohistochemistry staining --- p.31<br>Chapter 2. 1. 1. 4 --- Reagents and equipment for flow cytometry --- p.32<br>Chapter 2. 1. 2 --- Buffer --- p.32<br>Chapter 2. 1. 2. 1 --- Buffers for immunohistochemistry and immunofluorescence staining --- p.32<br>Chapter 2. 1. 2. 2 --- Buffers for western blot --- p.35<br>Chapter 2. 1. 3 --- Sequences of primers for genotyping and real-time PCR --- p.41<br>Chapter 2. 1. 4 --- Antibodies --- p.42<br>Chapter 2. 2 --- Methods --- p.44<br>Chapter 2. 2. 1 --- Generation of gene modified mice --- p.44<br>Chapter 2. 2. 2 --- Bone marrow transplantation --- p.45<br>Chapter 2. 2. 3 --- Conditional macrophage deletion --- p.45<br>Chapter 2. 2. 4 --- Unilateral ureteral obstruction (UUO) mouse model --- p.46<br>Chapter 2. 2. 5 --- Histology and immunohistochemistry --- p.46<br>Chapter 2. 2. 5. 1 --- Processing paraffin sections --- p.46<br>Chapter 2. 2. 5. 2 --- Deparaffinization and hydration --- p.47<br>Chapter 2. 2. 5. 3 --- Blocking endogenous peroxidase --- p.47<br>Chapter 2. 2. 5. 4 --- Antigen retrieval --- p.48<br>Chapter 2. 2. 5. 5 --- Antigen and antibody reaction --- p.48<br>Chapter 2. 2. 5. 6 --- Detection of target signals --- p.49<br>Chapter 2. 2. 5. 7 --- Quantification of immunohistochemistry staining --- p.49<br>Chapter 2. 2. 6 --- Immunofluorescence staining and confocal microscopy analysis --- p.49<br>Chapter 2. 2. 6. 1 --- Processing tissue for immune-fluorescent (IF) staining --- p.49<br>Chapter 2. 2. 6. 2 --- Serum blocking --- p.50<br>Chapter 2. 2. 6. 3 --- Antigen antibody reaction --- p.50<br>Chapter 2. 2. 6. 4 --- Signal detection --- p.51<br>Chapter 2. 2. 7 --- Flow cytometry --- p.52<br>Chapter 2. 2. 7. 1 --- Preparation of single cell suspension --- p.52<br>Chapter 2. 2. 7. 2 --- Cell fixation and permeabilization --- p.53<br>Chapter 2. 2. 7. 3 --- Staining --- p.53<br>Chapter 2. 2. 7. 4 --- Signal detection and analysis --- p.54<br>Chapter 2. 2 .8 --- Real time PCR --- p.55<br>Chapter 2. 2. 8. 1 --- Total RNA extraction --- p.55<br>Chapter 2. 2. 8. 2 --- Reverse transcription --- p.56<br>Chapter 2. 2. 8. 3 --- Real-time PCR --- p.57<br>Chapter 2. 2. 8. 4 --- Analysis of real-time PCR --- p.57<br>Chapter 2. 2. 9 --- Western blot --- p.58<br>Chapter 2. 2. 9. 1 --- Protein extraction from tissue --- p.58<br>Chapter 2. 2. 9. 2 --- Protein concentration measurement --- p.59<br>Chapter 2. 2. 9. 3 --- SDS-PAGE electrophoresis --- p.59<br>Chapter 2. 2. 9. 4 --- Protein transfer --- p.60<br>Chapter 2. 2. 9. 5 --- Blocking --- p.61<br>Chapter 2. 2. 9. 6 --- Antibodies incubation and signal detection --- p.62<br>Chapter 2. 2. 9. 7 --- Stripping --- p.62<br>Chapter CHAPTER III --- p.63<br>EVIDENCE FOR MMT AS A NEW PATHWAY OF MYOFIBROBLAST ORIGIN IN RENAL FIBROSIS --- p.63<br>Chapter 3. 1 --- Introduction --- p.64<br>Chapter 3. 2 --- Materials and methods --- p.65<br>Chapter 3. 2. 1 --- Human renal biopsy tissues --- p.65<br>Chapter 3. 2. 2 --- Experimental design --- p.65<br>Chapter 3. 2. 3 --- Bone marrow transplantation and GFP⁺ BM chimeric mice --- p.66<br>Chapter 3. 2. 4 --- Immunohistochemistry --- p.66<br>Chapter 3. 2. 5 --- Immunofluorescence and confocal microscopy analysis --- p.67<br>Chapter 3. 2. 6 --- Real-time PCR --- p.68<br>Chapter 3. 2. 7 --- Western blot analysis --- p.68<br>Chapter 3. 2. 8 --- Flow cytometry --- p.68<br>Chapter 3. 3 --- Results --- p.69<br>Chapter 3. 3. 1 --- BM-derived myofibroblasts play a key role in renal fibrosis in a mouse model of UUO --- p.69<br>Chapter 3. 3. 1. 1 --- α-SMA⁺ myofibroblasts are derived from BM and determine renal fibrosis in a mouse model of UUO --- p.69<br>Chapter 3. 3. 1. 2 --- BM as a major source of collagen production in a mouse model of UUO --- p.73<br>Chapter 3. 3. --- 2 Evidence for BM derived macrophage-myofibrobalst transition (MMT) in a mouse model of UUO --- p.77<br>Chapter 3. 3. 2. 1 --- Characterization of GFP⁺ BM chimeric mice --- p.77<br>Chapter 3. 3. 2. 2 --- Evidence for bone marrow-derived MMT is the major source of myofibroblast origin in the UUO kidney --- p.79<br>Chapter 3. 3. 3 --- Evidence for MMT in human fibrotic kidney tissues --- p.84<br>Chapter 3. 3. 4 --- M2 macrophage is the predomimant phenotype of macrophages in the fibrotic kidney of UUO mouse model. --- p.88<br>Chapter 3. 4 --- Discussion --- p.90<br>Chapter 3. 5 --- Conclusion --- p.93<br>Chapter CHAPTER IV --- p.94<br>Chapter GE --- CONDITIONAL MACROPHA DELETION INHIBITS MMT AND RENAL FIBROSIS --- p.94<br>Chapter 4. 1 --- Introduction --- p.95<br>Chapter 4. 2 --- Materials and methods --- p.98<br>Chapter 4. 2. 1 --- Generation of lysM-Cre/DTR mice --- p.98<br>Chapter 4. 2. 2 --- Conditional deletion of macrophage --- p.98<br>Chapter 4. 2. 3 --- Unilateral Ureteral Obstruction (UUO) mouse model --- p.98<br>Chapter 4. 2. 4 --- Real-time PCR --- p.99<br>Chapter 4. 2. 5 --- Western blot analysis --- p.99<br>Chapter 4. 2. 6 --- Immunohistochemisty --- p.99<br>Chapter 4. 2. 7 --- Immunofluorescence --- p.99<br>Chapter 4. 3 --- Results --- p.100<br>Chapter 4. 3. 1 --- Characterization of lysM-Cre/DTR mice --- p.100<br>Chapter 4. 3. 2 --- Conditional deletion of macrophage in a mouse model of UUO --- p.101<br>Chapter 4. 3. 3 --- Conditional deletion of macrophage suppresses α-SMA⁺ myofibroblast accumulation in a mouse model of UUO --- p.104<br>Chapter 4. 3. 4 --- Conditional deletion of macrophage inhibits collagen I production in a mouse model of UUO --- p.106<br>Chapter 4. 3. 5 --- Conditional deletion of macrophage inhibits renal fibrosis through reducing MMT cells in a mouse model of UUO --- p.108<br>Chapter 4. 4 --- Discussion --- p.111<br>Chapter 4. 5 --- Conclusion --- p.113<br>Chapter CHAPTER V --- p.114<br>MMT CELLS SHARE PERICYTE AND FIBROCYTE PHENOTYPES --- p.114<br>Chapter 5. 1 --- Introduciton --- p.115<br>Chapter 5. 2 --- Materials and methods --- p.116<br>Chapter 5. 2. 1 --- Human renal biopsy tissues --- p.116<br>Chapter 5. 2. 2 --- Animals and UUO mouse model --- p.116<br>Chapter 5. 2. 3 --- Immunofluorescence (IF) --- p.116<br>Chapter 5. 2. 4 --- Flow cytometry --- p.117<br>Chapter 5. 3 --- Results --- p.119<br>Chapter 5. 3. 1 --- Evidence for MMT cells co-expressing pericyte marker in the fibrotic kidney of UUO model --- p.119<br>Chapter 5. 3. 2 --- Evidence for MMT cells co-expressing pericyte marker in the fibrotic kidney from patients with chronic kidney diseases --- p.124<br>Chapter 5. 3. 3 --- Evidence for MMT cells co-expressing fibrocyte marker in the fibrotic kidney of UUO model --- p.126<br>Chapter 5. 3. 4 --- Evidence for MMT cells co-expressing fibrocyte marker in the fibrotic kidney from patients with chronic kidney diseases --- p.129<br>Chapter 5. 4 --- Dscussion --- p.131<br>Chapter 5. 5 --- Conclusion --- p.133<br>Chapter CHAPTER VI --- p.134<br>SMAD3 MEDIATES MMT DURING RENAL FIBROSIS --- p.134<br>Chapter 6. 1 --- Introduction --- p.135<br>Chapter 6. 2 --- Materials and methods --- p.137<br>Chapter 6. 2. 1 --- Generation of Smad3⁺/⁺ and Smad3⁻/⁻ BM-Chimeric mice --- p.137<br>Chapter 6. 2. 2 --- Generation of TbRII disrupted BM macrophages and Smad3⁻/⁻ BM macrophages --- p.137<br>Chapter 6. 2. 3 --- UUO mouse model --- p.138<br>Chapter 6. 2. 4 --- Cell culture --- p.138<br>Chapter 6. 2. 5 --- Real-time PCR --- p.139<br>Chapter 6. 2. 6 --- Western blot analysis --- p.139<br>Chapter 6. 2. 7 --- Immunohistochemistry (IHC) --- p.139<br>Chapter 6. 2. 8 --- Immunofluorescence (IF) --- p.139<br>Chapter 6. 2. 9 --- Flow cytometry --- p.140<br>Chapter 6. 3 --- Result --- p.141<br>Chapter 6. 3. 1 --- Genotyping of Smad3 WT and Smad3 KO mice --- p.141<br>Chapter 6. 3. 2 --- Smad3 knockout inhibits TGF-β1 induced MMT in vitro --- p.142<br>Chapter 6. 3. 3 --- Disruption of TbRII inhibits TGF-β1 induced MMT in vitro --- p.145<br>Chapter 6. 3. 4 --- Deletion of BM Smad3 inhibits α-SMA expression in the UUO kidney --- p.147<br>Chapter 6. 3. 5 --- Deletion of BM Smad3 inhibits collagen-I production in the UUO kidney --- p.149<br>Chapter 6. 3. 6 --- Inhibition of MMT is a mechanism by which BM Smad3 deficiency inhibits renal fibrosis in a mouse model of UUO --- p.150<br>Chapter 6. 4 --- Discussion --- p.153<br>Chapter 6. 5 --- Conclusion --- p.154<br>Chapter CHAPTER VII --- p.155<br>SUMMARY AND DISCUSSION OF THE MAJOR FINDINGS --- p.155<br>Chapter 7. 1 --- Summary and discussion --- p.157<br>Chapter 7. 1. 1 --- MMT is a major pathway of myofibroblast origin in renal fibrosis --- p.157<br>Chapter 7. 1. 2 --- MMT cells shares both pericyte and fibrocyte phenotypes in renal fibrosis --- p.157<br>Chapter 7. 1. 3 --- TGF-β/Smad3 is a key mechanism of MMT in renal fibrosis --- p.158<br>Chapter 7. 2 --- Conclusion --- p.160<br>Chapter REFERENCES --- p.161

Indiana University-Purdue University Indianapolis (IUPUI)<br>The Cre/loxP system is a tool for targeted recombination of DNA. For applying Cre recombinase-mediated genome modifications, there is a requirement for reliable, high-fidelity, and specific transgenic expression of the Cre recombinase. This study focuses on the reliability of different bone cell specific Cre models in the Cre/loxP system. In this study, DMP1-Cre transgenic mouse which has a transgene driven by DMP1 promotor that allows Cre-expression only in late stage osteoblasts and osteocytes was used. Ctsk-Cre mouse with a driven by Ctsk promoter was used so that only osteoclasts would undergo Cre-mediated recombination. E2A-Cre mouse where the Cre recombinase is driven by a global promoter E2A was also included in this study as a control line to test the Cre reporter line Ai9. Dmp1-Cre, Ctsk-Cre and E2A-Cre mice were crossed to the fluorescent Cre-reporter line—Ai9, which harbors a floxed stop codon, followed by the fluorophoremTomato, inserted into the Rosa26 locus. This construct is expected to give red fluorescence when it recombines with Cre-expressing mouse cells and no fluorescence in non-recombinant mouse cells. Double positive (Ai9+/Cre+) offspring selected by PCR were perfused, and 5mu-m thick section of bone and soft tissues were examined for red fluorescent expression. Cre positive cells were quantitated using ‘ImageJ’ software program. The DMP1-vi Cre mouse results showed significant expression in the targeted osteocytes and osteoblasts. In addition, skeletal muscle tissue also showed significant Cre- expression. Ctsk-Cre mice showed significant expression in targeted osteoclasts. But brain tissue was positive in Cre-expression. Bone-Cre mouse models are expected to express Cre recombinase only in their respective bone cells and they have been used for gene deletion studies in bone cells. However, this study has revealed that the bone cell specific Cre mouse models DMP1-Cre and Ctsk-Cre have unexpected expression in muscle and brain respectively. In order to use these models for targeted gene deletion in bone cells, further testing and studies have to be conducted.

Meng, Fanbiao.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.<br>Includes bibliographical references (leaves 114-138).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.

Indiana University-Purdue University Indianapolis (IUPUI)<br>Skeletal manifestations including short stature, osteoporosis, kyphoscoliosis, and tibial dysplasia cumulatively affect approximately 70% of patients with neurofibromatosis type 1 (NF1). Tibial pseudarthrosis, the chronic non-union of a spontaneous fracture, is a debilitating skeletal malady affecting young children with NF1. These non-healing fractures respond poorly to treatment and often require amputation of the affected limb due to limited understanding of the causative mechanisms. To better understand the cellular and molecular pathogenesis of these osseous defects, we have established a new mouse model which recapitulates a spectrum of skeletal pathologies frequently observed in patients with NF1. Nf1flox/-;Col2.3Cre mice, harboring Nf1 nullizygous osteoblasts on a Nf1+/- background, exhibit multiple osseous defects which are closely reminiscent of those found in NF1 patients, including runting (short stature), bone mass deficits, spinal deformities, and tibial fracture non-union. Through adoptive bone marrow transfer studies, we have demonstrated that the Nf1 haploinsufficient hematopoietic system pivotally mediates the pathogenesis of bone loss and fracture non-union in Nf1flox/-;Col2.3Cre mice. By genetic ablation of a single Nf1 allele in early myeloid development, under the control of LysMCre, we have further delineated that Nf1 haploinsufficient myeloid progenitors and osteoclasts are the culprit lineages mediating accelerated bone loss. Interestingly, conditional Nf1 haploinsufficiency in mature osteoclasts, induced by CtskCre, was insufficient to trigger enhanced lytic activity. These data provide direct genetic evidence for Nf1’s temporal significance as a gatekeeper of the osteoclast progenitor pool in primitive myelopoiesis. On the molecular level, we found that transforming growth factor-beta1 (TGF-β1), a primary mediator in the spatiotemporal coupling of bone remodeling, is pathologically overexpressed by five- to six- fold in both NF1 patients and in mice. Nf1 deficient osteoblasts, the principal source of TGF-β1 in the bone matrix, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, p21Ras dependent hyperactivation of the Smad pathway accentuates responses to pathological TGF-β1 signals in Nf1 deficient bone cells. As a proof of concept, we demonstrate that pharmacologic TβRI kinase inhibition can rescue bone mass defects and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice, suggesting that targeting TGF-β1 signaling in myeloid lineages may provide therapeutic benefit for treating NF1 skeletal defects.

前言：間充質幹細胞容易擴增並且能分化為成骨細胞、軟骨細胞和脂肪細胞，並且能對炎症、感染和損傷做出反應，並且遷移到相應的組織部位。這些特性使間充質幹細胞成為骨骼組織工程學中非常重要的細胞來源。外周血間充質幹細胞是一種存在於血液中的間充質幹細胞，而主要的間充質幹細胞存在與骨髓中，被稱之為骨髓間充質幹細胞。在我們實驗室之前的研究中通過DNA微陣列發現外周血間充質幹細胞中很多基因的表達與骨髓間充質幹細胞有很大區別。這其中的一些基因可能參與調控間充質幹細胞的分化和歸巢，我們從中挑選了三個變化比較明顯的基因--CRBP1, N-cadherin和 SOX11做進一步研究。本研究的目的在於研究CRBP1, N-cadherin和 SOX11在骨髓間充質幹細胞分化和遷移中的作用及相關機理。<br>方法：培養的骨髓間充質幹細胞來源於6-8周大小的SD大鼠。細胞的表型經過多分化潛能測試（成骨分化，成脂分化和成軟骨分化）和流式細胞儀檢驗。克隆大鼠的CRBP1, N-cadherin和SOX11基因到慢病毒載體。而且還設計了針對CRBP1和 N-cadherin的shRNA及非特異性對照shRNA。慢病毒由暫態轉染293FT細胞產生。細胞遷移實驗採用了BD Falcon的細胞遷移系統（cell culture insert）。實驗採用了定量PCR、免疫共沉澱、western雜交和雙螢光報告檢驗。對於體內實驗，細胞經感染帶有不同基因的病毒後，種植到Si-TCP材料並移植到裸鼠皮下。8周後，收集樣品進行組織學和免疫組織學分析。最後，我們建立了大鼠的股骨開放式骨折模型，並在4天后將SOX11基因修飾的間充質幹細胞通過心臟注射打到大鼠體內。4周後，收集股骨骨折樣品並進行microCT、力學測試和組織學分析。<br>結果：CRBP1過表達能夠促進骨髓間充質幹細胞的成骨分化潛能，並能抑制其成脂分化。進一步的機理研究表明CRBP1可以通過與RXRα的蛋白相互作用抑制RXRα誘導的β-catenin降解，從而維持β-catenin和磷酸化-ERK1/2在較高的水準，導致間充質幹細胞成骨能力增強；N-cadherin過表達可以促進間充質幹細胞的遷移，但是卻通過下調β-catenin和磷酸化ERK1/2抑制其成骨分化。過表達SOX11可以通過增強BMP信號通路促進三系分化。SOX11還可以通過啟動CXCR4的表達來促進細胞遷移。最後，在大鼠的股骨開放骨折模型上通過系統注射，我們證明穩定過表達SOX11的間充質幹細胞遷移到骨折部位的數量明顯增加。這些細胞到達骨折部位以後可以起始骨痂的鈣化，促進骨折的修復。<br>結論：本研究證明CRBP1, N-cadherin 和SOX11具有調節骨髓間充質幹細胞遷移和/或分化的功能。這些基因也許會成為幹細胞治療的新靶點。系統注射SOX11基因修飾的骨髓間充質幹細胞對於骨折修復可能具有較好的療效。本研究初步研究了CRBP1, N-cadherin 和SOX11在間充質幹細胞中的作用，為探討以間充質幹細胞為基礎的組織工程的某些新臨床應用提供了一些線索。<br>Introduction: Mesenchymal stem cells (MSCs) can be easily harvested, expanded, and have the capability of differentiating into osteoblasts, chondrocytes and adipocytes, and they can home to various tissues in response to stimuli such as inflammation, infection and injuries. MSCs are therefore valuable cell source for musculoskeletal tissue engineering. Peripheral blood-derived MSCs (PB-MSCs) are one kind of MSCs that reside in peripheral blood, whereas the main source of MSCs is bone marrow-derived MSCs (BM-MSCs). In our previous study, we found many genes were differentially expressed in the PB-MSCs compared to their counterpart BM-MSCs demonstrated by microarray analysis, among which the effects of CRBP1, SOX11 and N-cadherin on MSCs in terms of migration and differentiation are studied.<br>Methods: BM-MSCs and PB-MSCs were cultured from 6-8 weeks SD rats. The phenotypes of MSCs were characterized by tri-lineage (adipo-, osteo- and chondrogenic) differentiation and flow cytometry analysis. The genes encoding rat CRBP1, SOX11 and N-cadherin were cloned into lentiviral vectors respectively. shRNAs targeting CRBP1, N-cadherin, and one nonspecific shRNA were designed. Pseudo-lentivirus was produced by transient transfection of 293FT cells. Cell migration was examined using transwell insert culture system. Quantitative RT-PCR, CO-IP, western blot and dual-luciferase assay were employed in the studies. For in vivo study, MSCs transduced with different genes were seeded on Si-TCP scaffolds and implanted subcutaneously in nude mice. 8 weeks later, the samples were collected for histological and immunohistological analysis. Finally, an open femoral fracture model was established in 8-week old SD rats, SOX11-modified MSCs were injected at four days after fracture. At 4-week after MSCs injection, the femurs were collected for microCT, mechanical test and histological analysis.<br>Results: For CRBP1gene, our results showed that CRBP1 overexpression promoted osteogenic differentiation of BM-MSCs, while inhibited their adipogenic differentiation. We demonstrated that CRBP1 promoted osteogenic differentiation by inhibiting RXRα-induced β-catenin degradation through physical interactions, and maintaining β-catenin and pERK1/2 at higher levels. For N-cadherin gene, we found that N-cadherin overexpression promoted MSCs migration, and suppressed osteogenic potential of MSCs through inhibiting ERK and β-catenin signaling pathways. For SOX11 gene, we demonstrated that SOX11 overexpression enhanced the adipo-, osteo- and chondrogenic differentiation of BM-MSCs, through enhancing BMP signaling pathways. The migration capacity of BM-MSCs was also enhanced when Sox-11 was overexpressed, through activating CXCR4 expression. Finally, in the open femur fracture model we demonstrated that a larger number of SOX11-overexpressing BM-MSCs migrated to the fracture site, initiated earlier callus ossification and improved bone fracture healing quality.<br>Conclusions: This study demonstrated that CRBP1, N-cadherin and SOX11 gene can regulate the migration and/or differentiation potentials of BM-MSCs. These genes may become new therapeutic targets in stem cell therapy applications. Systemic administration of genetically modified SOX11-overexpressing BM-MSCs may be useful in promoting fracture healing. Overall, this study defined some unknown functions of CRBP1, N-cadherin and SOX11 in MSCs and shed the lights on some novel therapeutic implications for MSCs-based tissue engineering.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Xu, Liangliang.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 128-144).<br>Abstract also in Chinese.<br>Declaration --- p.i<br>Abstract --- p.ii<br>摘要 --- p.v<br>Acknowledgements --- p.vii<br>Chapter 1 --- p.1<br>Introduction --- p.1<br>Chapter 1.1 --- Mesenchymal stem cells --- p.2<br>Chapter 1.1.1 --- Characteristics of mesenchymal stem cells --- p.2<br>Chapter 1.1.2 --- Bone marrow- and peripheral blood-derived MSCs --- p.4<br>Chapter 1.1.3 --- Other tissue-derived MSCs --- p.5<br>Chapter 1.2 --- Adipogenesis of MSCs --- p.6<br>Chapter 1.3 --- Chondrogenesis of MSCs --- p.7<br>Chapter 1.4 --- Osteogenesis of MSCs --- p.8<br>Chapter 1.4.1 --- Regulators of osteogenesis --- p.9<br>Chapter 1.4.2 --- Stratergies for improving bone tissue engineering --- p.11<br>Chapter 1.5 --- Signaling pathways involved in osteogenesis --- p.13<br>Chapter 1.5.1 --- ERK signaling pathway --- p.14<br>Chapter 1.5.2 --- Wnt signaling pathway --- p.15<br>Chapter 1.5.3 --- BMP signaling pathway --- p.17<br>Chapter 1.6 --- Migration of MSCs --- p.20<br>Chapter 1.7 --- Fracture healing --- p.22<br>Chapter 1.8 --- Clinical application of MSCs --- p.23<br>Chapter 1.8.1 --- BM-MSCs vs. PB-MSCs --- p.24<br>Chapter 1.8.2 --- Autologous vs. Allogeneic MSCs transplantation --- p.25<br>Chapter 1.9 --- Scope of the present study --- p.26<br>Chapter 1.9.1 --- CRBP1 --- p.26<br>Chapter 1.9.2 --- N-cadherin --- p.27<br>Chapter 1.9.3 --- SOX11 --- p.27<br>Chapter 1.10 --- Experimental scheme --- p.29<br>Chapter 2 --- p.31<br>Comparison between PB-MSCs and BM-MSCs --- p.31<br>Chapter 2.1 --- Chapter introduction --- p.32<br>Chapter 2.2 --- Materials and methods --- p.33<br>Chapter 2.2.1 --- Cell culture --- p.33<br>Chapter 2.2.2 --- Flow cytometry --- p.33<br>Chapter 2.2.3 --- Adipogenic differentiation --- p.34<br>Chapter 2.2.4 --- Osteogenic differentiation --- p.34<br>Chapter 2.2.5 --- RNA Extraction and Real-time PCR --- p.34<br>Chapter 2.3 --- Results --- p.35<br>Chapter 2.3.1 --- Morphology of PB-MSCs --- p.35<br>Chapter 2.3.2 --- Cellular surface markers of BM-MSCs and PB-MSCs --- p.36<br>Chapter 2.3.3 --- Multi-differentiation potential of BM-MSCs and PB-MSCs --- p.38<br>Chapter 2.3.4 --- Target genes expression in BM-MSCs and PB-MSCs --- p.39<br>Chapter 2.4 --- Discussion and future work --- p.40<br>Chapter 3 --- p.41<br>Role of CRBP1 in Differentiation and Migration of MSCs --- p.41<br>Chapter 3.1 --- Chapter introduction --- p.42<br>Chapter 3.2 --- Materials and methods --- p.46<br>Chapter 3.2.1 --- Chemicals --- p.46<br>Chapter 3.2.2 --- Isolation and culture of BM-MSCs --- p.46<br>Chapter 3.2.3 --- RNA Extraction and Real-time PCR --- p.47<br>Chapter 3.2.4 --- Plasmid construction, transfection, production of lentivirus and infection --- p.48<br>Chapter 3.2.5 --- Osteogenic differentiation --- p.50<br>Chapter 3.2.6 --- Adipogenic differentiation --- p.50<br>Chapter 3.2.7 --- Western blot --- p.51<br>Chapter 3.2.8 --- Immunofluorescence labeling and fluorescence microscopy --- p.52<br>Chapter 3.2.9 --- Cell migration assay --- p.52<br>Chapter 3.2.10 --- Ectopic bone formation assay --- p.52<br>Chapter 3.2.11 --- Statistical analysis --- p.53<br>Chapter 3.3 --- Results --- p.53<br>Chapter 3.3.1 --- Transducing BM-MSCs with lentivirus carrying CRBP1 or shRNAs --- p.53<br>Chapter 3.3.2 --- CRBP1 accelerates osteogenesis of BM-MSCs via enhancing ERK1/2 and β-catenin pathways --- p.56<br>Chapter 3.3.3 --- CRBP1 stabilizes β-catenin by inhibiting RXRα-induced degradation --- p.58<br>Chapter 3.3.4 --- CRBP1 inhibits adipogenesis of BM-MSCs --- p.61<br>Chapter 3.3.5 --- CRBP1 overexpression has no effect on MSCs migration potential --- p.63<br>Chapter 3.3.6 --- CRBP1 promotes ectopic bone formation in vivo --- p.64<br>Chapter 3.4 --- Discussion --- p.66<br>Chapter 3.5 --- Future work --- p.73<br>Chapter 4 --- p.74<br>Role of N-cadherin in Differentiation and Migration of MSCs --- p.74<br>Chapter 4.1 --- Chapter introduction --- p.75<br>Chapter 4.2 --- Materials and methods --- p.78<br>Chapter 4.2.1 --- Chemicals --- p.78<br>Chapter 4.2.2 --- Isolation and culture of BM-MSCs --- p.78<br>Chapter 4.2.3 --- Plasmid construction, transfection, production of lentivirus and infection --- p.79<br>Chapter 4.2.4 --- Osteogenic differentiation and ALP activity assay --- p.81<br>Chapter 4.2.5 --- Western blot --- p.81<br>Chapter 4.2.6 --- Ectopic bone formation assay --- p.82<br>Chapter 4.2.7 --- Statistical analysis --- p.82<br>Chapter 4.3 --- Results --- p.83<br>Chapter 4.3.1 --- Expression of N-cadherin during osteogenesis in MSCs --- p.83<br>Chapter 4.3.2 --- N-cadherin overexpression inhibits osteogenesis through suppressing β-catein and ERK1/2 signaling pathways --- p.84<br>Chapter 4.3.3 --- N-cadherin silencing increases osteogenesis through enhancing β-catenin and ERK1/2 signaling pathways --- p.86<br>Chapter 4.3.4 --- N-cadherin promotes migration of MSCs --- p.87<br>Chapter 4.3.5 --- Cellular surface markers of SV40-immortalized MSCs --- p.89<br>Chapter 4.3.6 --- N-cadherin inhibits ectopic bone formation in vivo --- p.89<br>Chapter 4.4 --- Discussion --- p.91<br>Chapter 4.5 --- Future work --- p.94<br>Chapter 5 --- p.96<br>Role of SOX11 in Differentiation and Migration of MSCs --- p.96<br>Chapter 5.1 --- Chapter introduction --- p.97<br>Chapter 5.2 --- Materials and methods --- p.105<br>Chapter 5.2.1 --- Plasmid construction, transfection, production of lentivirus and infection --- p.105<br>Chapter 5.2.2 --- Cell culture --- p.106<br>Chapter 5.2.3 --- Luciferase reporter gene assay --- p.106<br>Chapter 5.2.4 --- Osteogenic differentiation and ALP activity assay --- p.106<br>Chapter 5.2.5 --- Adipogenic differentiation --- p.107<br>Chapter 5.2.5 --- Chondrogenic diffferentiation --- p.107<br>Chapter 5.2.6 --- Western blot --- p.108<br>Chapter 5.2.7 --- RNA Extraction and Real-time PCR --- p.108<br>Chapter 5.2.8 --- Cell migration --- p.110<br>Chapter 5.2.9 --- Ectopic bone formation --- p.110<br>Chapter 5.2.10 --- Fracture healing model and analysis --- p.111<br>Chapter 5.2.11 --- Statistical Analysis --- p.112<br>Chapter 5.3 --- Results --- p.112<br>Chapter 5.3.1 --- SOX11 is upregulated during osteogenesis of BM-MSCs --- p.112<br>Chapter 5.3.2 --- SOX11 promotes adipogenesis in BM-MSCs --- p.113<br>Chapter 5.3.3 --- SOX11 promotes migration of BM-MSCs --- p.114<br>Chapter 5.3.4 --- SOX11 promotes osteogenesis in BM-MSCs --- p.115<br>Chapter 5.3.5 --- SOX11 promotes chondrogenesis of MSCs --- p.117<br>Chapter 5.3.6 --- Mechanisms of how SOX11 regulates differentiation and migration of MSCs --- p.118<br>Chapter 5.3.7 --- SOX11-modified MSCs promote bone fracture healing in an open femur fracture rat model --- p.122<br>Chapter 5.4 --- Discussion --- p.126<br>Chapter 5.5 --- Future work --- p.131<br>Appendix --- p.153

Indiana University-Purdue University Indianapolis (IUPUI)<br>Signal Transducer and Activator of Transcription 3 (Stat3) is a transcription factor expressed in bone and joint cells that include osteoblasts, osteocytes, osteoclasts, and chondrocytes. Stat3 is activated by a variety of cytokines and growth factors, including IL-6/gp130 family cytokines. These cytokines not only regulate the differentiation of osteoblasts and osteoclasts, but also regulate proliferation of chondrocytes through Stat3 activation. In 2007, mutations of Stat3 have been confirmed to cause a rare human immunodeficiency disease – Job syndrome which presents skeletal abnormalities like: reduced bone density (osteopenia), scoliosis, hyperextensibility of joints, and recurrent pathological bone fractures. Changes in the Stat3 gene alter the structure and function of the Stat3 proteins, impairing its ability to control the activity of other genes. However, little is known about the effects of Stat3 mutations on bone cells and tissues. To investigate the in vivo physiological role of Stat3 in bone homeostasis, osteoblast/osteocyte-specific Stat3 knockout (KO) mice were generated via the Cre-LoxP recombination system. The osteoblast/osteocyte-specific Stat3 KO mice showed bone abnormalities and an osteoporotic phenotype because of a reduced bone formation rate. Furthermore, inactivation of Stat3 decreased load-driven bone formation, and the disruption of Stat3 in osteoblasts suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Stat3 has been found to be responsive to mechanical stimulation, and might play an important role in mechanical signal transduction in osteocytes. To investigate the role Stat3 plays in mechanical signaling transduction, osteocyte-specific Stat3 knockout (KO) mice were created. Inactivation of Stat3 in osteocytes presented a significantly reduced load-driven bone formation. Decreased osteoblast activity indicated by reduced osteoid surface was also found in osteocyte-specific Stat3 KO mice. Moreover, sclerostin (SOST) protein which is a critical osteocyte-specific inhibitor of bone formation, its encoded gene SOST expression has been found to be enhanced in osteocyte-specific Stat3 KO mice. Thus, these results clearly demonstrated that Stat3 plays an important role in bone homeostasis and mechanotransduction, and Stat3 is not only involved in bone-formation-important genes regulation in the nucleus but also in mediation of ROS and oxidative stress in mitochondria.

Indiana University-Purdue University Indianapolis (IUPUI)<br>The PRL family of enzymes constitutes a unique class of protein tyrosine phosphatase, consisting of three highly homologous members (PRL-1, PRL-2, and PRL-3). Family member PRL-3 is highly expressed in a number of tumor types and has recently gained much interest as a potential prognostic indicator of increased disease aggressiveness and poor clinical outcome for multiple human cancers. PRL-1 and PRL-2 are also known to promote a malignant phenotype in vitro, however, prior to the present study, little was known about their expression in human normal or tumor tissues. In addition, the biological function of all three PRL enzymes remains elusive and the underlying mechanisms by which they exert their effects are poorly understood. The current project was undertaken to expand our knowledge surrounding the normal cellular function of the PRL enzymes, the signaling pathways in which they operate, and the roles they play in the progression of human disease. We first characterized the tissue distribution and cell-type specific localization of PRL-1 and PRL-2 transcripts in a variety of normal and diseased human tissues using in situ hybridization. In normal, adult human tissues we found that PRL-1 and PRL-2 messages were almost ubiquitously expressed. Only highly specialized cell types, such as fibrocartilage cells, the taste buds of the tongue, and select neural cells displayed little to no expression of either transcript. In almost every other tissue and cell type examined, PRL-2 was expressed strongly while PRL-1 expression levels were variable. Each transcript was widely expressed in both proliferating and quiescent cells indicating that different tissues or cell types may display a unique physiological response to these genes. In support of this idea, we found alterations of PRL-1 and PRL-2 transcript levels in tumor samples to be highly tissue-type specific. PRL-1 expression was significantly increased in 100% of hepatocellular and gastric carcinomas, but significantly decreased in 100% of ovarian, 80% of breast, and 75% of lung tumors as compared to matched normal tissues from the same subjects. Likewise, PRL-2 expression was significantly higher in 100% of hepatocellular carcinomas, yet significantly lower in 54% of kidney carcinomas compared to matched normal specimens. PRL-1 expression was found to be associated with tumor grade in the prostate, ovary, and uterus, with patient gender in the bladder, and with patient age in the brain and skeletal muscle. These results suggest an important, but pleiotropic role for PRL-1 and PRL-2 in both normal tissue function and in the neoplastic process. These molecules may have a tumor promoting effect in some tissue types, but inhibit tumor formation or growth in others. To further elucidate the signaling pathways in which the PRLs operate, we focused on PRL-1 and used microarray and microRNA gene expression profiling to examine the global molecular changes that occur in response to stable PRL-1 overexpression in HEK293 cells. This analysis led to identification of several molecules not previously associated with PRL signaling, but whose expression was significantly altered by exogenous PRL-1 expression. In particular, Filamin A, RhoGDIalpha, and SPARC are attractive targets for novel mediators of PRL-1 function. We also found that PRL-1 has the capacity to indirectly influence the expression of target genes through regulation of microRNA levels and we provide evidence supporting previous observations suggesting that PRL-1 promotes cell proliferation, survival, migration, invasion, and metastasis by influencing multi-functional molecules, such as the Rho GTPases, that have essential roles in regulation of the cell cycle, cytoskeletal reorganization, and transcription factor function. The combined results of these studies have expanded our current understanding of the expression and function of the PRL family of enzymes as well as of the role these important signaling molecules play in the progression of human disease.

A population of stromal cells that retains osteogenic capacity in adult bone (adult bone stromal cells or aBSCs) exists and is under intense investigation in relation to osteogenesis and relevant pathology. aBSCs may be different from their embryonic or neonatal counterparts, and are influenced by species-/age-specific and other factors. Mice heterozygous for a null allele of prkar1a (Prkar1a+/-, a gene encoding for cyclic adenosine mono-phosphate (cAMP)-dependent regulatory subunit of protein kinase A (PKA), developed bone lesions that resembled fibrous dysplasia (FD) originated from cAMP-responsive osteogenic cells. Prkar1a +/- mice were crossed with mice heterozygous for catalytic subunit Calpha (Prkaca+/-), the main PKA activity-mediating molecule and generated mouse model with double heterozygosity for prkar1a and prkaca (Prkar1a +/-Prkaca+/-). Unexpectedly, Prkar1a+/-Prkaca+/- mice developed a large number of osseous lesions starting at 2--3 months of age that varied from the rare chondromas in the long bones and the ubiquitous osteochondrodysplasia of tail vertebral bodies to the occasional sarcoma in older animals. Cells from these lesions were fibroblast- and FD-like, and almost always originated from an area proximal to the growth plate and adjacent to endosteal surface of the periosteum; they expanded gradually in the bone marrow space. These cells expressed osteogenic cell markers, showed higher PKA activity that was mostly type II (PKA-II) and display an alternate pattern of catalytic subunit expression, and surprisingly possessed higher cAMP levels. In addition, markers of bone synthesis and lysis were increased. Gene expression profiling not only confirmed an early (progenitor) osteoblastic nature for these cells but also showed a signature that was indicative of mesenchymal-to-epithelial (MET) transition and increased Wnt signaling, particularly the brachyury expression. These studies show that a specific subpopulation of aBSCs can be stimulated in adult bone by PKA-II and altered Calpha activity, generating the only available germline mutant mouse model of a disorder that has similarities to human FD. Along with previous data, these studies also suggest that the effects of cAMP signaling on osteogenesis and stromal cell maintenance and proliferation in mice are age-, bone-, site- but also PKA-type and catalytic subunit-specific.<br>Parts of the work have been published in Proceedings of the National Academy of Sciences of the United States of America 2010; 107(19):8683--8.<br>Tsang, Kit Man.<br>Advisers: Constantine A. Stratakas; Kwak-Pui Fung.<br>Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: .<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.<br>Includes bibliographical references (leaves 144-183).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.

Indiana University-Purdue University Indianapolis (IUPUI)<br>Dicalcium phosphate dihydrate (DCPD) cements have been used for bone repair due to its excellent biocompatibility and resorbability. However, DCPD cements are typically weak and brittle. To overcome these limitations, the sodium citrate used as a setting regulator and the coating of poly-L-lactide acid (PLLA) technique have been proposed in this study. The first purpose of this thesis is to develop composite PLLA/DCPD scaffolds with enhanced toughness by PLLA coating. The second purpose is to examine the biocompatibility of the scaffolds. The final purpose is to investigate the degradation behaviors of DCPD and PLLA/DCPD scaffolds. In this experiment, DCPD cements were synthesized from monocalcium phosphate monohydrate (MCPM) and 𝛽-tricalcium phosphate (𝛽 –TCP) by using deionized water and sodium citrate as liquid components. The samples were prepared with powder to liquid ratio (P/L) at 1.00, 1.25 and 1.50. To fabricate the PLLA/DCPD composite samples, DCPD samples were coated with 5 % PLLA. The samples were characterized mechanical properties, such as porosity, diametral tensile strength, and fracture energy. The mechanical properties of DCPD scaffolds with and without PLLA coating after the in vitro static degradation (day 1, week1, 4, and 6) and in vitro dynamic degradation (day 1, week 1, 2, 4, 6, and 8) were investigated by measuring their weight loss, fracture energy, and pH of phosphate buffer solution. In addition, the dog bone marrow stromal stem cells (dBMSCs) adhesion on DCPD and PLLA/DCPD composite samples were examined by scanning electron microscopy. The cell proliferation and differentiation in the medium conditioned with DCPD and PLLA/DCPD composite samples were studied by XTT (2,3-Bis(2-methoxy-4- nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt), and alkaline phosphatase (ALP) assay, respectively. The addition of sodium citrate and PLLA coating played a crucial role in improving the mechanical properties of the samples by increasing the diametral tensile strength from 0.50 ± 0.15 MPa to 2.70 ± 0.54 MPa and increasing the fracture energy from 0.76 ± 0.18 N-mm to 12.67 ± 4.97 N-mm. The DCPD and PLLA/DCPD composite samples were compatible with dBMSCs and the cells were able to proliferate and differentiate in the conditioned medium. The degradation rate of DCPD and PLLA/DCPD samples were not significant different (p > 0.05). However, the DCPD and PLLA/DCPD composite samples those used sodium citrate as a liquid component was found to degrade faster than the groups that use deionized water as liquid component

Indiana University-Purdue University Indianapolis (IUPUI)<br>Gain-of-function mutations in the KIT receptor tyrosine kinase have been associated with highly malignant human neoplasms. In particular, an acquired somatic mutation at codon 816 in the second catalytic domain of KIT involving an aspartic acid to valine substitution is found in patients with systemic mastocytosis (SM) and acute myeloid leukemia (AML). The presence of this mutation in SM and AML is associated with poor prognosis and overall survival. This mutation changes the conformation of the KIT receptor resulting in altered substrate recognition and constitutive tyrosine autophosphorylation leading to constitutive ligand independent growth. As there are currently no efficacious therapeutic agents against this mutation, this study sought to define novel therapeutic targets that contribute to aberrant signaling downstream from KITD816V that promote transformation of primary hematopoietic stem/progenitor cells in diseases such as AML and SM. This study shows that oncogenic KITD814V (murine homolog) induced myeloproliferative neoplasms (MPN) occurs in the absence of ligand stimulation, and that intracellular tyrosines are important for KITD814V-induced MPN. Among the seven intracellular tyrosines examined, tyrosine 719 alone has a unique role in regulating KITD814V-induced proliferation and survival. Residue tyrosine 719 is vital for activation of the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), p85α, downstream from KITD814V. Downstream effectors of the PI3K signaling pathway, in of leukemic cells bearing KITD814V with an allosteric inhibitor of Pak or its genetic inactivation results in growth repression due to enhanced apoptosis. To assess the role of Rac GEFs in KITD814V induced transformation, EHop-016, an inhibitor of Rac, was used to specifically target Vav1, and found to be a potent inhibitor of human and murine leukemic cell growth. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of MPN and rescued the associated pathology in mice. These studies provide insight on mechanisms and potential novel therapeutic targets for hematological malignancies harboring an oncogenic KIT mutation.