Effects of brefeldin-A: Potent inhibitor of intracellular protein transport on ultrastructure and resorptive function of cultured osteoclasts

Brefeldin-A (BFA) is a specific and potent inhibitor of the intracellular t ransport of clathlin-uncoated transitional vesicles from the cisterns of ro ugh-surfaced endoplasmic reticulum (RER) to the Golgi lamellae. This study was designed to clarify the effects of BFA on ultrastructure, subcellular l ocalization of vacuolar-type H+-ATPase and a lysosomal cysteine proteinase, cathepsin K, in cultured osteoclasts and their resorptive function. H+-ATP ase and cathepsin K are the most important enzymes for decalcification of a patite crystals and degradation of type-I collagen, respectively. In contro l cultures without BFA, osteoclasts were structurally characterized by the development of broad ruffled borders and clear zones, and formed many resor ption lacunae in cocultured dentine slices. In BFA-treated cultures, osteoc lasts lacked ruffled borders, and the cytoplasm was filled with regular-siz e and extremely large pale vacuoles over 2 mum in diameter, which were prod uced by fusion of adjacent vacuoles. BFA did not, however, inhibit clear zo ne formation and adhesion of osteoclasts to dentine slices. Resorption lacu na formation was markedly diminished by BFA treatment. Although H+-ATPase a nd cathepsin K were strongly expressed in osteoclast ruffled borders in con trol cultures, BFA treatment altered the subcellular localization and decre ased the expression of these molecules. In BFA-treated cultures, H+-ATPase immunoreaction in osteoclasts was observed along the limiting membranes of some, but not all, regular-size pale vacuoles, but neither in extremely lar ge vacuoles nor along the smooth plasma membranes facing the dentine slices . Similarly, cathepsin K was localized within lysosomes and some regular-si ze pale vacuoles, but its secretion toward the dentine slices through the r uffled borders was strongly inhibited by BFA treatment. These results sugge st that 1.) formation of the osteoclast ruffled borders and their resorptiv e function are closely associated with the intracellular transport of these molecules from the RER cisterns and the Golgi lamellae to the ruffled bord ers, and 2.) both H+-ATPase and cathepsin K are selectively transported to the ruffled border membranes by pale vacuoles. Anat Rec 263:127-138, 2001. (C) 2001 Wiley-Liss, Inc.