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  Cytoskeleton modification and the effect on acid secretion in osteoclasts
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 Ophav:
Junker, Märta Caroline1, Forfatter
Klærke, Dan Arne1, Vejleder
Henriksen, Kim1, Vejleder
Tilknytninger:
1Det Biovidenskabelige Fakultet, København, Danmark
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 Abstract: The osteoclast is a bone cell which is involved in osteoporosis and it is the only cell type in the human body with the ability to degrade bone, a process known as bone resorption. Normally, the osteoclasts are tightly regulated and involved in the continuing bone remodelling necessary for maintaining a healthy skeleton. In healthy individuals, bone remodelling, where bone is resorbed by osteoclasts and new bone is formed by osteoblasts, is tightly coupled in the sense that bone resorption equals bone formation. During bone disease, e.g. osteoporosis, bone resorption exceeds the bone formation resulting in fragile bone and bone loss leading to increased fracture risk. The osteoclast is multinuclear, highly polarized and has an actin ring, unique to this cell type. During bone resorption the osteoclast attaches itself to bone and secretes hydrochloric acid, HCl, in an extracellular compartment, the resorption lacuna, towards the bone surface, dissolving the mineralized bone. HCl is secreted via the vacuolar ATPase, vATPase, and a chloride transporter, ClC-7, both present in the plasma membrane termed “the ruffled border” in osteoclasts.Inhibiting osteoclastic bone resorption by disruption of the actin ring may be a target for inhibition of acidification and thus bone resorption. Compounds modifying the cytoskeleton of cells may be used to study actin ring disruption in osteoclasts. In this project five known cytoskeleton modulators were tested in a lysosomal acidification assay, using osteoclasts formed in vitro by isolating CD14+ monocytes from human blood and differentiating them to osteoclasts in the presence of M-CSF and RANKL. One compound, U18666A, inhibited lysosomal acidification significantly. U18666A was tested further in a membrane vesicle acid influx assay, a vATPase assay, actin ring staining and in a bone resorption assay. U18666A inhibited acidification, vATPase activity and bone resorption but phalloidin-TRITC staining of the actin rings showed no actin ring disruption. U18666A was toxic in high concentrations and its inhibition of acidification could be due to it disrupting cell membranes.Osteoclasts possess a G protein coupled receptor, the calcitonin receptor (CTR), which has been shown to induce actin ring disruption upon ligand binding, thus inhibiting bone resorption. Calcitonin is a peptide hormone, which binds the CTR, and is an approved pharmaceutical in treatment for osteoporosis and other bone diseases affected by osteoclasts. Calcitonin and SUNB8155, a small molecule CTR agonist, were tested in a cAMP assay, to test their ability to induce cAMP, a known secondary messenger in a CTR intracellular signalling pathway. Calcitonin, but not SUNB8155, induced cAMP formation in osteoclasts. The compounds were also tested for cAMP formation in a CTR overexpressing cell line, COS-7-CTR+ cells and both calcitonin and SUNB8155 gave a cAMP response. SUNB8155 was not as potent as calcitonin and did not induce as high cAMP response as calcitonin in the COS-7-CTR+ cells.Calcitonin and SUNB8155 were also tested for actin ring disruption and inhibition of bone resorption. Calcitonin disrupted actin rings of osteoclasts and inhibited bone resorption, while SUNB8155 proved to be toxic in high concentrations and no conclusions could be made on its ability to mimic calcitonins effects in osteoclasts.The disruption of the actin ring of osteoclasts is a potential target for inhibition of bone resorption.The test for inhibition of the acidification of osteoclasts when exposed to known cytoskeleton modifiers did not succeed in finding a molecule which inhibited both acidification and the osteoclastic cytoskeleton. A small molecule CTR agonist may be a treatment for osteoporosis, and for finding such a molecule, the setup of good screening assays validating the effect on CTR function is essential. In this report such assays were validated with the natural CTR agonist, calcitonin.
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CJU_BTK08014.pdf (Hovedtekst)
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Bogmærk denne post: https://diskurs.kb.dk/item/diskurs:12333:1
 Type: Speciale
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Sprog: English - eng
 Datoer: 2010
 Sider: 80 pages
 Publiceringsinfo: København, Danmark : Institut for Basal Husdyr- og Veterinærvidenskab
 Indholdsfortegnelse: -
 Note: Biology - Biotechnology, Biologi - Bioteknologi
 Type: Speciale
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