Open AccessSpecific biological functions of vacuolar‐type H + ‐ATPase and lysosomal cysteine proteinase, cathepsin K, in osteoclasts
Author(s) -
Sahara Takako,
Itoh Kanami,
Debari Kazuhiro,
Sasaki Takahisa
Publication year - 2003
Publication title -
the anatomical record part a: discoveries in molecular, cellular, and evolutionary biology
Language(s) - English
Resource type - Journals
eISSN - 1552-4892
pISSN - 1552-4884
DOI - 10.1002/ar.a.10020
Subject(s) - bafilomycin , osteoclast , cathepsin , resorption , bone resorption , v atpase , cathepsin k , chemistry , microbiology and biotechnology , cytoplasm , atpase , ultrastructure , multinucleate , cathepsin l , biochemistry , biology , enzyme , anatomy , endocrinology , in vitro , apoptosis , autophagy
We report the effects of specific and potent inhibitors of vacular‐type H + ‐ATPase and lysosomal cysteine proteinases, cathepsins, on the ultrastructure, expression of these enzymes, and resorptive functions of cultured osteoclasts. Osteoclasts were formed by co‐culture of marrow cells and calvarial primary osteoblasts of ddY mice. Formed osteoclasts were cultured on dentine slices for 6–48 hr with either an H + ‐ATPase inhibitor, bafilomycin A1, or a cysteine proteinase inhibitor, E‐64. In control cultures with no additive, osteoclasts were structurally characterized by the development of ruffled borders and clear zones, and formed many resorption lacunae on dentine slices. Both H + ‐ATPase and cathepsin K were strongly expressed in the ruffled borders of these osteoclasts. In bafilomycin A1‐treated cultures, osteoclasts lacked ruffled borders, and resorption lacuna formation was markedly diminished. This effect of bafilomycin A1 on osteoclast structure was reversible by removal of the compound. Bafilomycin A1 treatment altered the subcellular localization and decreased the expression of H + ‐ATPase molecules. H + ‐ATPase expression was observed throughout the cytoplasm, but not along the plasma membranes facing dentine slices. On the other hand, E‐64 treatment did not affect the ultrastructure of osteoclasts and the expression of enzyme molecules. Although E‐64 showed no effect on demineralization of dentine slices, it dose‐dependently reduced resorption lacuna formation. Our results suggest that 1) bafilomycin A1 dose‐dependently inhibits resorption lacuna formation via inhibition of ruffled border formation, 2) H + ‐ATPase expression is closely associated with the cytoskeleton of osteoclasts, and 3) E‐64 treatment decreases the depth of resorption lacunae, by inhibition of secreted cathepsin K activity, but does not impair ruffled border formation and the associated expression of H + ‐ATPase and cathepsin K in osteoclasts. Anat Rec Part A 270A:152–161, 2003. © 2003 Wiley‐Liss, Inc.