Thesis (Ph.D.)--Aberdeen University, 2009. / Title from web page (viewed on Oct. 8, 2009). Includes bibliographical references.

Thesis (Ph.D.)--Aberdeen University, 2009. / Title from web page (viewed on Jan. 5, 2010). Includes bibliographical references.

The osteoclast ruffled border is a highly convoluted, complex membrane that is necessary for bone resorption. It is thought to form following mass lysosomal fusion with the boneapposing plasma membrane and vesicular trafficking is vital for its formation and function. The aim of this PhD was to better understand the ultrastructure, formation and function of the ruffled border using TEM and advanced imaging techniques. Ruffled border reformation following calcitonin treatment was visualised and the stages of ruffled border formation were described. Ruffled borders in healthy and osteopetrotic osteoclasts were also imaged by TEM and characterised using a morphological grading system. The key findings of this thesis are as follows: (1) vacuoles, not lysosomes, are the primary contributors of membrane to the ruffled border and the membrane projections of the ruffled border form passively as a consequence of channel formation, not actively by membrane folding, (2) extracellular vesicles are located, and appear to be released, at the ruffled border. Various functional aspects of the ruffled border were also investigated. Vesicles near the ruffled border were identified and characterised by immunoelectron microscopy based on their content and morphology. We found no morphological defects in ruffled borders in mice deficient in Plekhm1. In osteoclasts derived from patients with a SNX10 mutation, we found that while the cells retained the capacity to form well-developed ruffled borders, they did so less often than healthy control osteoclasts. Importantly, we observed that even in a population of healthy osteoclasts, ruffled border morphology is highly heterogeneous because they are at different stages in the resorption cycle. In conclusion, the data in this thesis provide novel findings, previously unseen details regarding how resorbing osteoclasts interact with the bone surface, and have revealed unique insights into ruffled border morphology, formation and the vesicles with which it interacts.

Rheumatoid arthritis (RA) is a systemic, inflammatory disease, which predominantly affects multiple joints. RA is characterized by swollen joints and peri-articular bone erosion. Conventional RA treatments have shown to reduce inflammation and slow down bone erosion, but repair of bone erosion is limited. Additionally, failure to repair for bone erosion in rheumatoid arthritis (RA) is caused by inadequate osteoblast-mediated bone formation resulting from focal inflammatory environment. Hence, there is immediate need to facilitate greater insight and develop a new therapeutic strategy to aid osteoblast -mediated repair of bone loss in RA. CKIP-1 is an intracellular inhibitor, that can negatively regulate osteoblast lineage cells differentiation and activity. CKIP-1 levels were found to be aberrantly over expressed in bone specimens from RA patients and arthritis mice, which was associated with reduced bone formation and increased disease severity. By genetic approach, overexpressed CKIP-1 in osteoblast exacerbated bone erosion and articular inflammation in CIA mice, whereas deficiency of CKIP-1 in osteoblast alleviates bone erosion in CIA mice. By pharmacological approach, RNA interference-based silencing of osteoblastic CKIP-1 led to bone formation-mediated reparative process at erosive site and reduced articular inflammation in arthritis induced mice. To extend above findings to a more relevant species that more closely resemble humans, we aimed to investigate the role of osteoblastic Casein kinase-2 interacting protein-1 (CKIP-1) in failure to repair bone erosion in non-human primate (NHP) arthritis model in this study. We found that CKIP-1 mRNA expression in osteoblasts of arthritic NHP was significantly suppressed by CKIP-1 siRNA treatment. Moreover, silencing of CKIP-1 in osteoblast of arthritis monkey improved clinical signs such as reduction in arthritis score, joint swelling and increase in body weight. Similarly, suppression of osteoblastic CKIP-1 resulted in better organized bony and articular structure with, fewer bone erosion sites as observed in x ray and micro CT images. Moreover, we found increase in bone mass, bone formation parameters such as BFR/BS and MAR and histological examination revealed attenuation of peri articular bone erosion and intraarticular inflammation in CKIP-1 siRNA treated arthritis monkeys. Taken together, these data strongly suggest that highly expressed osteoblastic CKIP-1 plays an important role in failure to repair articular bone erosion by inhibiting bone formation in RA. Targeting osteoblastic CKIP-1 could serve as a new therapeutic strategy by bone repair augmentation in RA.

No description available.

Macrophages arise from two distinct lineages of hematopoietic cells, Hematopoietic Stem Cell-derived progenitors and Erythromyeloid Progenitors. EMP derived macrophages are seeded in tissues early during embryogenesis and become specialized tissue resident macrophages such as microglia and Kupffer cells. HSC derived monocytes arise in the bone marrow and normally circulate via the bloodstream to reach tissues of the body and differentiate into macrophages which are cells that are constantly replenished by their progenitors. Monocytes are also recruited to inflamed tissues attracted by inflammatory signals, where they become macrophages and further contributed to the inflammatory process. These two macrophage populations may be implicated in different activities and have different functions, such as tissue repair or proinflammatory responses, respectively. Other cells that are unique in having both origins are osteoclasts, which during embryogenesis originate from EMP precursors and contribute to the formation of ossification centers of long bones and consequently the formation of bone marrow cavity. After birth, HSC derived monocytes contribute to their cell maintenance by direct cell fusion to pre-existing EMP-derived osteoclasts, eventually replacing them to become fully derived from HSC precursors, a mechanism that takes several month to complete in mice. Cell lineage tracing is a powerful technique that allows for the labeling of specific cell populations in a specific time and space. The gold standard cell lineage tracing mouse model for the study of EMP derived cells including macrophages and osteoclasts in mice, Csf1rMeriCreMer;Rosa26LSL-YFP,1–3 uses a tamoxifen inducible Cre recombinase to induce expression of YFP and therefore permanently labels EMP progenitors and they progeny. Other mouse models to label macrophages and osteoclast have been used, which include Cx3cr1CreERT2;Rosa26LSL-tdTomato, however, recent evidence show spontaneous expression of tdTomato fluorescent protein in the absence of tamoxifen (unpulsed mice). Here we confirmed that Cre recombination resulted in tdTomato expression in the absence of tamoxifen in Cx3cr1CreERT2;Rosa26LSL-tdTomato mice. Therefore, we utilized a different fluorescent reporter gene instead, Rosa26LSL-YFP, and generated Cx3cr1CreERT2;Rosa26LSL-YFP mice for permanent labelling of EMP-derived cells, and provide evidence that no YFP expression is detected in unpulsed mice. Further, we performed pulse labeling of the Cx3cr1CreERT2;Rosa26LSL-YFP model at E10.5 days of gestation. These experiments were notable for YFP expression in macrophages and the highest labeling efficiency reported in osteoclasts (~70%) at E18.5 embryonic days of development. Further, there was no YFP expression detected in HSCs or HSC-derived cells, confirming this model as a potential useful tool for precise and efficient labelling of EMP-derived macrophages and osteoclasts

Bisphosphonates are the most widely prescribed anti-resorptive agents and work by preventing the post-translational modification (prenylation) of small GTPases in osteoclasts, subsequently leading to cell death by apoptosis.  Phosphonocarboxylate analogues of bisphosphonates also have anti-resorptive activity and work by inhibiting the enzyme Rab GGTase, thereby preventing the prenylation of Rab GTPases specifically.  Rab GTPases comprise a large family of related proteins that coordinate vesicular trafficking, which involves the processing, transportation and delivery of cellular cargo in a strict temporal and spatial manner.  In osteoclasts, vesicular trafficking is vital for the formation of the ruffled border (the resorptive organelle of the cell), the delivery of lytic enzymes and acid into the resorption space, and the uptake and disposal of bone degradation products.  However, the role that specific Rabs play in this functionally unique cell type remains poorly defined, and the Rab expression profile in osteoclasts is incomplete.  The work presented here aimed to increase our understanding of the role that Rabs play in osteoclasts.  Results indicate that the 70% reduction of Rab GGTase activity observed in <i>gunmetal </i>mice is detrimental to the activity of osteoclasts and osteoblasts <i>in vitro</i>, therefore highlighting the importance of Rabs for bone resorption and deposition.  Furthermore, this study is the first to determine the Rab expression profile of human osteoclasts, following a proteomic approach, and describes the transfection methods devised to characterise these candidate Rabs in osteoclasts.  Finally, this study details the characterisation of Rab18 in human osteoclasts, following its discovery during proteomic analysis.

Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

Recent studies have proposed that non-nitrogen-containing and nitrogen- containing bisphosphonate drugs inhibit osteoclastic bone resorption by different molecular mechanisms. The aim of this thesis was to investigate the molecular mechanisms of action of bisphosphonates in macrophages and osteoclasts and, in particular, the activation of caspase proteases and their role in apoptotic cell death. Apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates was found to involve the activation of caspase-3. By contrast, non-nitrogen- containing bisphosphonates did not cause caspase activation or J774 apoptosis, indicating that these bisphosphonates have different cellular effects. Further studies demonstrated that nitrogen-containing bisphosphonates induced J774 macrophage apoptosis by inhibiting the mevalonate pathway and preventing protein farnesylation and/or geranylgeranylation, since these compounds inhibited incorporation of [14 C] mevalonate into isoprenylated proteins, and addition of cell-permeable intermediates of the mevalonate pathway (FPP and GGPP) prevented bisphosphonate-induced apoptosis. Apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates or mevastatin (another inhibitor of the mevalonate pathway) was dependent on protein synthesis, since cycloheximide effectively prevented the activation of caspase-3 and prevented J774 cell apoptosis. Both nitrogen-containing bisphosphonates and non-nitrogen-containing bisphosphonates caused caspase-3 activation and apoptosis of rabbit and human osteoclasts in vitro. The active form of caspase-3 was detected in apoptotic osteoclasts by immunofluorescence staining, whilst caspase-3 activity was visualised in osteoclasts using a cell-permeable, fluorogenic substrate and detected in cell lysates using caspase-specific substrates. Bisphosphonate-induced osteoclast apoptosis involved loss of mitochondrial membrane potential and could be prevented by a specific inhibitor of caspase-3/-7. The ability of bisphosphonates to activate caspase-3 and cause apoptosis was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, suggesting that caspase activation and apoptosis in osteoclasts induced by bisphosphonates is the consequence of loss of geranylgeranylated proteins. Bisphosphonate-induced osteoclast apoptosis and inhibition of bone resorption in vitro was suppressed by RANK ligand. This did notappear to involve changes in Akt phosphorylation or increased expression of cIAP-1 or cIAP-2. These studies have helped to identify the molecular mechanisms of action of bisphosphonate drugs and have provided new insights into the involvement of caspases in osteoclast apoptosis.