Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains vi, 123 p. : ill. Includes abstract. Includes bibliographical references (p. 110-123).

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly upregulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and bone disease studies. Cellular organelles and vesicles in the exocytic and endocytic pathways have a distinctive spatial distribution and their trafficking is aided by many molecules, Rab GTPases being a master regulator. In this work, I identified the Rab GTPases that are upregulated during OB differentiation using microarray analysis, namely Rab1, Rab3d, and Rab27b, and investigated their role in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane (PM) utilizing their dominant negative (DN) expression. The experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels also was observed showing roles of Rab1, Rab3d and Rab27b in the specific function of these major collagen producing cells in the body. I further observed that a fraction of procollagen colocalized with lysosomes which was markedly increased when procollagen was experimentally misfolded. Lysosomes, essential organelles for intracellular degradation, are generally sequestered near the cell centre to receive vesicles with contents targeted for destruction. During AA-induced differentiation of OB cells, I saw a marked increase in total degradative lysosome organelles in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed toward the cell periphery in differentiating OBs without being secreted. This required intact microtubules for long range transport and was kinesin motor-dependent but did not involve cytosolic acidification. Moreover, impairment of lysosome dispersion markedly reduced AA-induced OB differentiation. Taken together, this study provides an important general mechanism for cell secretion phenomena that may ultimately lead to clinical targets for treatments of diseases driven by aberrant collagen processing and secretion including Osteogenesis Imperfecta (OI).

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly upregulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and bone disease studies. Cellular organelles and vesicles in the exocytic and endocytic pathways have a distinctive spatial distribution and their trafficking is aided by many molecules, Rab GTPases being a master regulator. In this work, I identified the Rab GTPases that are upregulated during OB differentiation using microarray analysis, namely Rab1, Rab3d, and Rab27b, and investigated their role in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane (PM) utilizing their dominant negative (DN) expression. The experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels also was observed showing roles of Rab1, Rab3d and Rab27b in the specific function of these major collagen producing cells in the body. I further observed that a fraction of procollagen colocalized with lysosomes which was markedly increased when procollagen was experimentally misfolded. Lysosomes, essential organelles for intracellular degradation, are generally sequestered near the cell centre to receive vesicles with contents targeted for destruction. During AA-induced differentiation of OB cells, I saw a marked increase in total degradative lysosome organelles in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed toward the cell periphery in differentiating OBs without being secreted. This required intact microtubules for long range transport and was kinesin motor-dependent but did not involve cytosolic acidification. Moreover, impairment of lysosome dispersion markedly reduced AA-induced OB differentiation. Taken together, this study provides an important general mechanism for cell secretion phenomena that may ultimately lead to clinical targets for treatments of diseases driven by aberrant collagen processing and secretion including Osteogenesis Imperfecta (OI).

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

published_or_final_version / Medicine / Master / Master of Philosophy

There is a growing need for bone cells to be used in the generation and repair of bone tissues that have become destroyed as a result of disease (e.g. osteoporosis), injuries and genetic deficiencies. Human umbilical cord blood has been a source of cells that is being investigated for its ability to generate cells for repair of various tissues including bone, which is the focus of this investigation. Similarly cord blood can also be used as a source of progenitors for osteoclasts that might be used for management of other diseases of bone characterized by defects in these cells (e.g. osteopetrosis). Moreover, there are few reliable in vitro models for human osteoclasts and so the ability to develop a cell model for human osteoclasts will also permit more meaningful studies on regulation of these cells than possible in the past. One of the setbacks suffered by researchers in designing therapeutic uses for stem cells is that in laboratory animals tumours can arise from these transplanted and pleuripotential cells. This suggests that stem cells might also have a dysregulated apoptosis pathway, which is critically important to understand before the safe use of stem cells can be assured. In order to increase our understanding of apoptosis in bone cells in particular, studies were done to try to understand the role of harakiri, a pro-apoptotic gene, in the development of osteoblasts and osteoclasts. Therefore, this series of study had two main foci; the development of human bone cells, particularly osteoclasts and osteoblasts, from human umbilical cord blood, and to understand further the mechanisms regulating apoptosis in bone cells. This study showed that osteoblasts could not be derived easily from human cord blood cells while it was possible to generate fully functional osteoclasts, which demonstrated unique properties. Studies done on harakiri deficient mice showed that absence of this gene caused an increase in osteoblast formation and a decrease in osteoclast formation. These findings can be exploited when considering the development of pharmacological agents that might be used in the future to modulate osteoclast cell development, function, and apoptosis.

There is a growing need for bone cells to be used in the generation and repair of bone tissues that have become destroyed as a result of disease (e.g. osteoporosis), injuries and genetic deficiencies. Human umbilical cord blood has been a source of cells that is being investigated for its ability to generate cells for repair of various tissues including bone, which is the focus of this investigation. Similarly cord blood can also be used as a source of progenitors for osteoclasts that might be used for management of other diseases of bone characterized by defects in these cells (e.g. osteopetrosis). Moreover, there are few reliable in vitro models for human osteoclasts and so the ability to develop a cell model for human osteoclasts will also permit more meaningful studies on regulation of these cells than possible in the past. One of the setbacks suffered by researchers in designing therapeutic uses for stem cells is that in laboratory animals tumours can arise from these transplanted and pleuripotential cells. This suggests that stem cells might also have a dysregulated apoptosis pathway, which is critically important to understand before the safe use of stem cells can be assured. In order to increase our understanding of apoptosis in bone cells in particular, studies were done to try to understand the role of harakiri, a pro-apoptotic gene, in the development of osteoblasts and osteoclasts. Therefore, this series of study had two main foci; the development of human bone cells, particularly osteoclasts and osteoblasts, from human umbilical cord blood, and to understand further the mechanisms regulating apoptosis in bone cells. This study showed that osteoblasts could not be derived easily from human cord blood cells while it was possible to generate fully functional osteoclasts, which demonstrated unique properties. Studies done on harakiri deficient mice showed that absence of this gene caused an increase in osteoblast formation and a decrease in osteoclast formation. These findings can be exploited when considering the development of pharmacological agents that might be used in the future to modulate osteoclast cell development, function, and apoptosis.

Bone remodelling, the process by which the skeleton adapts to environmental changes, is dependent on the actions of osteoclasts that resorb bone and osteoblasts which make new bone matrix. Aberrant remodelling underpins bone loss in several debilitating skeletal diseases such as osteoporosis, metastatic breast cancer and multiple myeloma. Changes in remodelling activity can also arise as a consequence of therapeutic intervention for instance intravenous bisphosphonate treatment is associated with osteochemonecrosis of the jaw and localised osteoradionecrosis is a common side effect of radiotherapy. Hyperbaric oxygen is often used as an adjunctive therapy in the treatment of these disorders. HBO involves the administration of 100% oxygen at atmospheric pressures greater than one in sealed chambers. The following studies aimed to evaluate the effect of HBO, hyperoxia, and pressure on RANKL-induced osteoclast differentiation and bone resorption from RAW264.7 and human peripheral blood mononuclear cells (PBMC), and osteoblast differentiation in vitro. The study also aimed to further examine the effect of HBO on ex vivo osteoclast formation from peripheral blood monocytes obtained from patients undergoing HBO. Daily exposure to HBO for ninety minutes significantly suppressed osteoclast differentiation and bone resorption in mouse and human monocytes in normoxic and hypoxic conditions in vitro. The suppressive action of HBO on osteoclast formation was associated with a significant reduction in HIF-1α and RANK mRNA expression and HBO also caused a significant reduction in NFATc1 and DC-STAMP expression. This study has for the first time shown that HBO is able to reduce the ability of precursors to form bone resorbing osteoclast. HBO also suppressed the ability of peripheral blood monocytes to develop into RANKL-induced resorptive osteoclasts. In an ex vivo culture system the suppressive effect of HBO was meditated by an action prior to activation of osteoclast differentiation by RANKL and must therefore be an inhibitory effect on the ability of precursors to differentiate along the osteoclastic lineage. HBO also accelerates the rate of osteoblast differentiation and augments early stages of mineralization and has a more pronounced effect than hyperoxia or pressure alone. HBO enhanced bone nodule formation and ALP activity in human osteoblasts. Furthermore HBO promoted the expression of type I collagen and Runx-2 in both normoxic and hypoxic conditions. HBO had a greater effect on these key markers of osteoblast differentiation than hyperoxia or pressure alone. This study suggests that HBO suppresses osteoclast activity and promotes osteoblastic bone formation, which may at least in part mediate its beneficial effects on necrotic bone. This provides evidence supporting the use of HBO as an adjunctive therapy to prevent osteoclast formation in a range of skeletal disorders associated with low oxygen partial pressure. The study also provides further support for the use of HBO in the treatment of skeletal disorders associated with excessive resorption such as osteomyelitis, and also provides a potential mechanism through which short term HBO may help fracture healing.

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Thesis (M.Med.Sc.)-- University of Adelaide, Dept. of Medicine, 2002. / Bibliography: leaves 86-103.