The proteinase responsible for bone collagen degradation in osteo-resorption was examined. The bone pit formation induced by parathyroid hormone (PTH) was markedly suppressed by leupeptin, E-64 and cystatin A, while no inhibition was observed by CA-074, a specific inhibitor of cathepsin B. Pig leucocyte cysteine proteinase inhibitor (PLCPI), a specific inhibitor of cathepsin L, and chymostatin, a selective inhibitor of cathepsin L, completely inhibited the pit formation. Cathepsin L activity in osteoclasts was much higher than the other cathepsin activities. Serum calcium in rats placed on a low calcium diet was decreased by treatment of E-64 or cystatin A, but not by CA-074. These findings suggest that cathepsin L is the main proteinase responsible for bone collagen degradation.

Secretion of procathepsin L into the culture medium from a bone cell mixture was markedly enhanced by addition of parathyroid hormone (PTH), 1 a,25-(OH)2D 3 or tumor necrosis factor a (TNFa). These stimulators of secretion of procathepsin L enhanced bone pit formation, which was inhibited by E-64, but not by CA-074, a specific inhibitor of cathepsin B. Procathepsin L may thus participate in the process of bone collagenolysis during bone resorption. Procathepsin L partially purified from rat long bones under cold conditions was rapidly converted to the mature form under acidic conditions at room temperature. This conversion was inhibited by E-64, suggesting that the procathepsin L secreted into lacunae is catalytically converted to the mature enzyme by cysteine proteinase(s).In the present study, designed to clarify the mechanism and regulation of both the secretion of procathepsin L and its processing in lacunae, we examined the secretion of procathepsin L induced by 10~,25-(OH)2D 3, TNFc~ or by PTH, and also the effects of the cysteine proteinase inhibitors, E-64 and CA-074, on the pit formation induced by these effectors. Furthermore, to clarify the participation of cysteine proteinase(s) in processing from the precursor to the active form, the inhibitory effect of E-64 on the processing of procathepsin L partially purified from rat long bones was also examined. Experimental

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The secretion mechanisms of cathepsin L from osteoclasts in the process of bone resorption were investigated. The increases in bone pit numbers formed take place by PTH addition in parallel with the increases of cathepsin L and/or L-like proteinase activities in the culture medium of bone cells, and these were suppressed by the addition of calcitonin. The Z-Phe-Arg-MCA hydrolysing activity increased in the medium through the effect of F'TH is considered to be a kind of procathepsin L by Western blotting analysis, and was suppressed by calcitonin addition. Furthermore, monensin inhibited not only the PTH-induced pit formation, but also cysteine proteinase activity in osteoclasts. Therefore, the procathepsin L excreted might be transferred from endothelial reticulum via Golgi and/or via lysosomes.

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