Quantification of the expression levels of lysosomal cysteine proteinases in purified human osteoclastic cells by competitive RT-PCR.
Cathepsin K is a lysosomal cysteine proteinase (LCP) predominantly expressed in osteoclasts. This study was conducted to evaluate the importance of human cathepsin K for osteoclastic bone resorption relative to that of other LCPs. To accomplish this, we quantitatively determined the expression levels of major LCPs (cathepsins B, K, L, and S) in human osteoclastic cells by using competitive RT-PCR. Giant cell tumor of bone (GCT) was used as a source of human osteoclastic cells, since the tissue was shown to contain a large number of cells satisfying the criteria for typical osteoclasts. The involvement of LCPs in the bone-resorption process by the GCT cells was confirmed by showing that trans-epoxysucciny-L-leucylamido-(4-guanidino) butane (E-64), a nonselective cysteine proteinase inhibitor, exerted an inhibitory effect on the pit formation. We isolated osteoclast-like cells (OLCs) positive for tartrate-resistant acid phosphatase (TRAP) and cathepsin K from the GCT tissue to a degree of almost 95% purity. In these cells, the expression of cathepsin K was shown to be approximately 20-, 130-, and 410-fold stronger than that of cathepsins B, L, and S, respectively. A similar result was obtained when human bone marrow cells in culture were used as another source of OLCs. Further, we found that cathepsin K was expressed in OLCs far more strongly than in several human nonosteoclastic cells including osteoblastic cell lines. The abundant and selective expression of cathepsin K in OLCs relative to that of other LCPs suggests that cathepsin K is mainly responsible for osteoclastic degradation of human bone matrix.
Ishibashi
O
O
Department of Dental Pharmacology, Niigata University School of Dentistry, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan.
Inui
T
T
Mori
Y
Y
Kurokawa
T
T
Kokubo
T
T
Kumegawa
M
M
D000135:Acid Phosphatase / Q000378:metabolism; D000328:Adult; D001854:Bone Marrow Cells / Q000187:drug effects / Q000201:enzymology; D001862:Bone Resorption / Q000201:enzymology / Q000517:prevention & control; D002116:Calcitonin / Q000494:pharmacology; D056657:Cathepsin K; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D018212:Giant Cell Tumor of Bone; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010006:Osteoblasts / Q000201:enzymology; D010010:Osteoclasts / Q000187:drug effects / Q000201:enzymology*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071681:Tartrate-Resistant Acid Phosphatase; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007527:Isoenzymes; D012333:RNA, Messenger; D002116:Calcitonin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534130:CTSK protein, human; D056657:Cathepsin K; D007930:Leucine; C024974:E 64