Osteoclast inhibitory peptide 2 inhibits osteoclast formation via its C-terminal fragment

Osteoclast inhibitory peptide 2 (OIP-2) is a novel autocrine/paracrine fact or produced by osteoclasts (OCLs) that inhibits bone resorption and OCL for mation in vitro and in vivo. It is identical to the asparaginyl endopeptida se legumain. During maturation of OIP-2, a signal peptide and a 17-kDa C-te rminal fragment (CTF) are cleaved to produce the mature enzyme. To determin e if enzyme activity is required for inhibition of OCL formation or if only the CTF is responsible for these effects, we synthesized His-tagged comple mentary DNA (cDNA) constructs for the CTF of OIP-2, the proform of OIP-2, a nd the "mature enzyme" form of OIP-2. The proform or the CTF portion of OIP -2 inhibited OCL formation in a dose-dependent manner in murine bone marrow cultures stimulated with 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3]. The m ature form of OIP-2, which was enzymatically active, did not inhibit OCL fo rmation. In addition, OIP-2 inhibited OCL formation in cultures of highly p urified human OCL precursor cells or RAW264.7 cells stimulated with 10 ng/m l of receptor activator of NF-kappaB (RANK) ligand. Binding studies with Hi s-tagged OIP-2 showed expression of a putative OIP-2 receptor on RAW264.7 c ells treated with RANK ligand for 4 days and human marrow cultures treated with 1,25(OH)(2)D-3 for 3 weeks. These data show that the CTF of OIP-2, rat her than the mature enzyme, mediates the inhibitory effects of OIP-2 throug h a putative receptor on OCL precursors.