Abstract
A precise control of vesicular trafficking is crucial not only for osteoclastic bone resorption, but also for the crosstalk between osteoclasts and osteoblasts, which regulates bone homeostasis. In addition to the release of growth factors and modulators, such as glutamate, flux through the intracellular trafficking routes could also provide the osteoclast with a monitoring function of its resorption activity. To establish the signaling pathways regulating trafficking events in resorbing osteoclasts, we used the bone conserving hormone calcitonin, which has the unique property of inducing osteoclast quiescence. Calcitonin acts through the calcitonin receptor and activates multiple signaling pathways. By monitoring trafficking of a fluorescent low molecular weight probe in mature, bone resorbing osteoclasts we show for the first time that calcitonin blocks endocytosis from the ruffled border by phospholipase C (PLC) activation. Furthermore, we identify a requirement for polyunsaturated fatty acids in endocytic trafficking in osteoclasts. Inhibition of PLC prior to calcitonin treatment restores endocytosis to 75% of untreated rates. This effect is independent of protein kinase C activation and can be mimicked by an increase in intracellular calcium. We thus define an essential role for intracellular calcium levels in the maintenance of endocytosis in osteoclasts.
Highlights
Bone homeostasis is a dynamic process, which relies on the crosstalk between bone forming osteoblasts, bone resident osteocytes, and bone resorbing osteoclasts (Matsuo, 2012; Xiong and O’Brien, 2012)
CALCITONIN AND cAMP ELEVATION INTERFERE WITH ENDOCYTOSIS IN ACTIVELY RESORBING OSTEOCLASTS In accordance with previously published results (Lucht, 1973; Baron et al, 1990), pre-incubation of rabbit osteoclasts plated on dentine slices with 20 nM calcitonin for 30 min markedly reduces the number of osteoclasts showing endocytosis compared to control samples (Figures 1A,B)
Our results show that forskolin has a more pronounced effect on the number of endocytosis-active osteoclasts than the cell-membrane permeable cAMP analogs, suggesting that the calcitonin effect on endocytosis is partially due to a combination of exchange-proteindirectly activated-by-cAMP (Epac)- and protein kinase A (PKA)-derived downstream signals
Summary
Bone homeostasis is a dynamic process, which relies on the crosstalk between bone forming osteoblasts, bone resident osteocytes, and bone resorbing osteoclasts (Matsuo, 2012; Xiong and O’Brien, 2012). Imbalances in the crosstalk between these cell types can lead to metabolic bone diseases such as osteoporosis and osteopetrosis (Teti, 2011). This important process is controlled by locally released factors as well as close cell interactions (Cao, 2011). Digested material is removed from the resorption pit by endocytosis and trafficked through the osteoclast via a transcytotic route to be released at the functional secretory domain at the bone avert side of the cell (Vaananen and Laitala-Leinonen, 2008). The extracellular calcium released during the resorption process is trafficked through the osteoclasts (Yamaki et al, 2005) and plays an important role in mediating osteoclast activity. It is likely that intracellular calcium levels play a role in regulating endocytosis and transcytosis in resorbing osteoclasts
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