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.