Cathepsin K (cat K) is the major
cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during
bone resorption. However, little is known regarding the synthesis, activation, or turnover of the endogenous
enzyme in osteoclasts. In this study, we show that mature cat K
protein and
enzyme activity are localized within osteoclasts. Pulse-chase experiments revealed that, following the synthesis of pro cat K, intracellular conversion to the mature
enzyme occurred in a time-dependent manner. Subsequently, the level of mature
enzyme decreased. Little or no cat K was observed in the
culture media at any timepoint. Pretreatment of osteoclasts with either
chloroquine or
monensin resulted in complete inhibition of the processing of newly synthesized cat K. In addition, pro cat K demonstrated susceptibility to treatment with
N-glycosidase F, suggesting the presence of high-
mannose-containing
oligosaccharides. Treatment of osteoclasts with the
PI3-kinase inhibitor,
Wortmannin (WT), not only prevented the intracellular processing of cat K but also resulted in the secretion of
proenzyme into the
culture media. Taken together, these results suggest that the biosynthesis, processing, and turnover of cat K in human osteoclasts is constitutive and occurs in a manner similar to that of other known
cysteine proteases. Furthermore, cat K is not secreted as a
proenzyme, but is processed intracellularly, presumably in lysosomal compartments prior to the release of active
enzyme into the resorption lacunae.