Characterization of Phagosomal Subpopulations along Endocytic Routes in Osteoclasts and Macrophages

Abstract

Modifications occurring during the transformation of phagosomes into mature phagolysosomes were investigated in osteoclast-like cells (OCLs) and macrophages using latex beads as markers for the isolation of phagosomal compartments (LBC) at different time points after phagocytosis. In OCLs, newly formed LBC acquired cathepsin K, tartarate-resistant phosphatase (TRAP), lysosome-associated membrane protein-1 (Lamp-1), and cathepsin D, and rapidly lost annexin II in a time-dependent manner. The levels of Rab7 and c-Src in OCLs initially increased and then gradually decreased during the transformation from early to late endosomal LBC or phagolysosomes. Receptor activator of NF-kappaB (RANKL) significantly increased the LBC levels of cathepsin K, TRAP, and c-Src, whereas calcitonin decreased the LBC levels of cathepsin K, TRAP, and Rab7, indicating that the transformation of early to late endosomal elements and lysosomes in OCLs is also regulated by osteoclastogenesis regulatory factors. On the other hand, changes in the LBC levels of Lamp-1, cathepsin D, and annexin II in macrophages were comparable to those in OCLs. However, contrary to osteoclastic LBC, Rab7 levels of macrophage LBC decreased in a time-dependent manner. Macrophage LBC were devoid of cathepsin K, TRAP, and c-Src in all transformation stages. These observations suggest that OCLs and macrophages have different phagosome maturation mechanisms that involve the specific and regulated acquisition of markers from endocytic organelles. The results also demonstrate that the use of LBC is a useful system in which to identify and characterize molecules involved in these different endocytic pathways.

Osteoactivin is a novel osteoclastic protein and plays a key role in osteoclast differentiation and activity

“…These findings suggest that OA resides in the endocytic pathway of osteoclasts and is probably targeted to the plasma membrane or extracellular space, where it exerts its biological function, upon osteoclast differentiation and maturation. In this regard, intracellular membrane trafficking and endocytic pathways are regulated by RANKL and are important for osteoclast function [28]. These findings are also consistent with our conclusion that OA is involved in primarily the late rather than early osteoclast differentiation processes.…”

Microphthalmia transcription factor regulates the expression of the novel osteoclast factor GPNMB

“…The localisation of GPNMB to these compartments is consistent with the presence of a predicted endosomal/ lysosomal-sorting signal located immediately after the transmembrane domain on the C terminal region of GPNMB (Anderson et al, 2002), (smart.embl-heidelberg.de). Intracellular membrane trafficking and endocytic pathways are regulated by the critical osteoclastogenic factor RANKL and are crucial for osteoclast function (Sakai et al, 2001). An important number of osteoclast molecules such as CTSK, CLCN7 and v-ATPase are transported from late endosomes/ lysosomes and recycling compartments to the extracellular space and ruffled border to carry out their function (Sahara, 2001;Toyomura et al, 2003;Lange et al, 2006).…”

“…6). Intracellular membrane trafficking and endocytic pathways are essential for osteoclast function and are regulated by factors such as RANKL (Sakai et al, 2001). Many other proteins that are enriched in osteoclasts such as TRAP, CTSK and CLCN7 are closely associated with these pathways and some of them are known to be transported from late endosomes/lysosomes or recycling compartments to the cell periphery, extracellular space and plasma membrane (ruffled border) (Sahara, 2001;Hollberg et al, 2002;Lange et al, 2006).…”

Annexin A2 Heterotetramer: Structure and Function

“…Interestingly, although AIIt is an actin bundling protein it does not play a role in stress fiber or filopodia formation. It is rather associated with dynamic membrane cytoskeletal structures such as rocketing macropinosomes [47] and phagosomes [113]. Thus, depletion of annexin A2 results in the enrichment of stress fibers and loss of dynamic ruffling physiology [112].…”