Cathepsin K is a recently identified cysteine protease which is abundantly and selectively expressed in osteoclasts. To evaluate the contribution of cathepsin K to bone resorption processes, we investigated the effect of cathepsin K antisense phosphothiorate oligodeoxynucleotide (S-ODN) on the bone-resorbing activity of osteoclasts. Rabbit osteoclasts were cultured on dentine slices for 24 h in the presence or absence of antisense S-ODN in a medium containing 100 nM Tfx TM -50, polycationic liposome, as a carrier of the S-ODN. Uptake of the S-ODN by osteoclasts was confirmed microscopically using fluorescein-labeled S-ODN. The treatment with antisense significantly decreased the amount of cathepsin K protein in osteoclasts. The antisense inhibited the osteoclastic pit formation in a concentration-dependent fashion. At 10 M the antisense reduced the total pit number and area and average pit depth by 46, 52, and 30%, respectively. The sense and mismatch SODNs, which were used as negative controls, had no effect on either the cathepsin K protein level or the pit formation. A nonspecific cysteine protease inhibitor, E-64, also reduced pit formation in a concentration-dependent manner with maximum reductions at 1 M of 46, 48, and 35% in the above pit parameters. The inhibitory effect of the antisense almost equal to that of E-64 demonstrates that cathepsin K is a cysteine protease playing a crucial role in osteoclastic bone resorption.Bone tissue is a composite matrix comprising of hydroxyapatite and fibrous proteins (mainly Type I collagen) and is constantly subjected to a cycle of bone resorption and bone formation (1). Bone resorption is mainly carried out by osteoclasts which are multinucleate giant cells. In osteoclastic bone resorption, demineralization, in which osteoclasts release protons to solubilize the inorganic salt (2), is followed by the degradation of the protein fibers with cysteine proteases (1, 3). The involvement of the cysteine proteases has been verified in both in vitro and in vivo studies showing that various types of cysteine protease inhibitors reduce bone resorption (1,(3)(4)(5)(6)(7)(8)(9)(10). From studies based on substrate preference, inhibitor preference, and immunoreactivity, the cathepsins L and B were suggested to be responsible for osteoclastic bone resorption processes (8 -14).Recently, several research laboratories (including our own) have successfully cloned cDNAs for a novel cysteine protease, namely cathepsin K, from rabbit and human cDNA libraries (15)(16)(17)(18)(19), and its role in bone resorption has been the focus of recent attention. Human cathepsin K is highly and selectively expressed in osteoclasts (16 -21); in fact its expression level is much greater than those of cathepsins B, L, and S (20, 21). Brömme et al. (22) and Bossard et al. (23) showed that cathepsin K expresses a potent proteolytic activity against Type I collagen. Saneshige et al. (24) demonstrated that retinoic acid, a vitamin A metabolite, both up-regulates the gene expression of cathepsin K in ost...