The N-terminal fragment of osteocalcin is released during osteoclastic bone resorption in vitro

Abstract

As a novel method for measuring bone resorption, an immunoassay system for human N-terminal osteocalcin (N-OC) was developed to determine if osteocalcin molecules are released from bone degraded by osteoclasts in vitro. The assay system employed a monoclonal antibody to the first 20 N-terminal residues of osteocalcin as the solid phase and polyclonal antibodies against these same residues as an enzyme conjugate. This assay system could detect the N-terminal portion of osteocalcin formed during the degradation of the human osteocalcin molecule by trypsin or cathepsin D. Osteoclasts were isolated from human alveolar bone and cultured on human bone slices; the osteocalcin content in the media from these cultures was measured by this N-OC assay method. The N-terminal fragment of osteocalcin was the major form of osteocalcin released during osteoclastic bone resorption along with small amounts of intact osteocalcin. Interleukin-1 (IL-1β) and interleukin-6 (IL-6), stimulators of osteoclastic bone resorption, increased the N-OC level by 1.5 fold compared with the level of N-OC in osteoclast cultures in the absence of stimulators. In contrast, treatment of the cultures with calcitonin or an inhibitor of cathepsin D (E-64), both of which inhibit osteoclastic bone resorption, decreased the amount of N-OC in the culture supernatants. These results suggest that the N-terminal fragment of osteocalcin is released during osteoclastic bone resorption and that its level may serve as an index of bone resorption in vitro.