title: Localization of rat cathepsin K in osteoclasts and resorption pits: inhibition of bone resorption and cathepsin K-activity by peptidyl vinyl sulfones.
abstract: We have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment. Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MbetaNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MbetaNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MbetaNA, was prevented by both types of inhibitors. The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.
authors.affiliation: Mount Sinai School of Medicine, Department of Human Genetics, New York, NY 10029, USA.
authors.forename: L
authors.initials: L
authors.lastname: Xia
authors.forename: J
authors.initials: J
authors.lastname: Kilb
authors.forename: H
authors.initials: H
authors.lastname: Wex
authors.forename: Z
authors.initials: Z
authors.lastname: Li
authors.forename: A
authors.initials: A
authors.lastname: Lipyansky
authors.forename: V
authors.initials: V
authors.lastname: Breuil
authors.forename: L
authors.initials: L
authors.lastname: Stein
authors.forename: J T
authors.initials: JT
authors.lastname: Palmer
authors.forename: D W
authors.initials: DW
authors.lastname: Dempster
authors.forename: D
authors.initials: D
authors.lastname: Brömme
mesh_terms: D000818:Animals; D001483:Base Sequence; D001862:Bone Resorption / Q000517:prevention & control*; D056657:Cathepsin K; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002451:Cell Compartmentation; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D017931:DNA Primers; D007150:Immunohistochemistry; D051379:Mice; D010010:Osteoclasts / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D011994:Recombinant Proteins; D013450:Sulfones; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K; C534132:Ctsk protein, mouse; C534133:Ctsk protein, rat