Identification of human asparaginyl endopeptidase (legumain) as an inhibitor of osteoclast formation and bone resorption

We screened a human osteoclast (OCL) cDNA expression library for OCL inhibi tory factors and identified a clone that blocked both human and murine OCL formation and bone resorption by more than 60%. This clone was identical to human legumain, a cysteine endopeptidase. Legumain significantly inhibited OCL-like multinucleated cell formation induced by 1,25-dihydroxyvitamin D- 3 (1,25-(OH)(2)D-3) and parathyroid hormone-related protein (PTHrP) in mous e and human bone marrow cultures, and bone resorption in the fetal rat long bone assay in a dose-dependent manner. Legumain was detected in freshly is olated marrow plasma from normal donors and conditioned media from human ma rrow cultures. Furthermore, treatment of human marrow cultures with an anti body to legumain induced OCL formation to levels that were as high as those induced by 1,25-(OH)(2)D-3. Implantation in nude mice of 293 cells transfe cted with the legumain cDNA and constitutively expressing high levels of th e protein significantly reduced hypercalcemia induced by PTHrP by about 50% , and significantly inhibited the increase in OCL surface and in OCL number expressed per mm(2) bone area and per mm bone surface induced by PTHrP. Th ese results suggest that legumain may be a physiologic local regulator of O CL activity that can negatively modulate OCL formation and activity.