Immunocytochemical study of cathepsin L and rat salivary cystatin-3 in rat osteoclasts treated with E-64 in vivo.
The localization of cathepsin L and rat salivary cystatin-3 (RSC-3) in rat osteoclasts (rat femoral and alveolar bones) treated with or without E-64 (control) was examined immunocytochemically. In osteoclasts pretreated with E-64, immunoreactivity for cathepsin L was very weak extracellularly compared to that in the control osteoclasts. However, it was strong intracellularly. The localization of RSC-3 was unclear in the control osteoclasts, while in E-64 treated osteoclasts, both the clear zone and ruffled border areas showed a very strong immunoreaction. At the electron-microscopic level, in normal osteoclasts, numerous immunoreaction products for cathepsin L were found extracellularly in the bone matrix under the ruffled border, while few intracellular products were observed. In contrast, in the E-64-treated osteoclasts, only a few immunoreaction products were found extracellularly, while intracellularly cathepsin L was found in numerous endosome-lysosomal vacuoles. In the immunoreaction for RSC-3, the cytoplasm of the ruffled border was positive, and the tips of the RSC-3-positive ruffled border appeared to enter deeply into the bone matrix. Intracellularly, the granular reaction products of RSC-3 were found in the vacuoles (probably autophagolysosomes). Thus, in E-64-treated osteoclasts, inhibition of the extracellular release of cathepsin L was demonstrated. In addition, intralysosomal accumulation of RSC-3 and deep penetration of the RSC-3-positive ruffled border into the bone matrix were found. These findings suggest that RSC-3 is associated with the inhibition of cathepsin L in both the lysosomes (in the osteoclasts) and bone matrix.
Moroi
R
R
Department of Prosthetic Dentistry I, Faculty of Dentistry, Kyushu University, Fukuoka, Japan.
Yamaza
T
T
Nishiura
T
T
Nishimura
Y
Y
Terada
Y
Y
Abe
K
K
Himeno
M
M
Tanaka
T
T
D000818:Animals; D001857:Bone Matrix / Q000737:chemistry / Q000201:enzymology; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D015891:Cystatins / Q000032:analysis* / Q000378:metabolism; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism* / Q000633:toxicity; D010450:Endopeptidases; D007930:Leucine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D016253:Microscopy, Immunoelectron; D010010:Osteoclasts / Q000737:chemistry* / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides / Q000032:analysis*
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534280:Ctsl protein, rat; D007930:Leucine; C024974:E 64