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).
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/33894 |
| Date | 06 December 2012 |
| Creators | Nabavi, Noushin |
| Contributors | Harrison, Rene E. |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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