Cathepsin B activity in normal human osteoblast-like cells and human osteoblastic osteosarcoma cells (MG-63): regulation by interleukin-1 beta and parathyroid hormone.
Cathepsin B activity and its regulation by interleukin 1 beta (IL-1 beta) and parathyroid hormone (PTH) was investigated in normal human osteoblast-like cells (hOB) and in the human osteoblastic osteosarcoma cell line MG-63. Cathepsin B activity was measured using a fluorescent synthetic substrate, 7-N-benzyloxycarbonyl-L-arginyl-L-arginylamide-4-methylcoumarin, and its specificity was checked with E-64, a specific inhibitor of cysteine proteinases and CA074, a specific inhibitor of the enzyme. Cathepsin B activity was detected in crude extracts of cell monolayers and in conditioned media. In both cell types, basal activity was detected essentially in cell extracts, since in media only approximately 1.2% (hOB) and approximately 6% (MG-63) of the total activity was released. IL-1 beta (1-100 U/ml) and PTH (10(-9) M-10(-6) M) significantly stimulated cathepsin B activity in cell extracts and in conditioned media. In both cell types, the increase in proteolytic activity appeared to require RNA and protein synthesis after adding IL-1 beta or PTH. Using the above substrate, we also evaluated some biochemical properties of the enzyme, and its pH-stability and pH-optimum. In both cell types, intracellular cathepsin B activity was not resistant to neutral or slightly alkaline pH, whereas extracellular cathepsin B activity was stable. This study provides evidence that osteoblast-like cells produce and secrete active cathepsin B. The production and secretion was stimulated by IL-1 beta and PTH. The physiological role of cathepsin B produced by osteoblasts and stimulated by the bone resorbing agents remains to be elucidated. Since extracellular activity is stable under relatively physiological conditions, it is possible that the extracellular as well as intracellular form of the enzyme may play a role in matrix turnover.
Aisa
M C
MC
Department of Human Metabolism and Clinical Biochemistry, University of Sheffield, Medical School, UK.
Rahman
S
S
Senin
U
U
Maggio
D
D
Russell
R G
RG
D002401:Cathepsin B / Q000378:metabolism*; D003374:Coumarins / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry; D004789:Enzyme Activation; D004795:Enzyme Stability; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007375:Interleukin-1 / Q000494:pharmacology*; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology*; D012516:Osteosarcoma / Q000201:enzymology* / Q000473:pathology; D010281:Parathyroid Hormone / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D003374:Coumarins; D004151:Dipeptides; D005456:Fluorescent Dyes; D007375:Interleukin-1; D010281:Parathyroid Hormone; C069012:benzyloxycarbonylarginyl-arginine 4-methylcoumarin-7-ylamide; D002401:Cathepsin B