Osteoclast Inhibitory Peptide 2 Inhibits Osteoclast Formation via Its C‐Terminal Fragment

Osteoclast inhibitory peptide 2 (OIP‐2) is a novel autocrine/paracrine factor produced by osteoclasts (OCLs) that inhibits bone resorption and OCL formation in vitro and in vivo. It is identical to the asparaginyl endopeptidase legumain. During maturation of OIP‐2, a signal peptide and a 17‐kDa C‐terminal fragment (CTF) are cleaved to produce the mature enzyme. To determine if enzyme activity is required for inhibition of OCL formation or if only the CTF is responsible for these effects, we synthesized His‐tagged complementary DNA (cDNA) constructs for the CTF of OIP‐2, the proform of OIP‐2, and 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 mature form of OIP‐2, which was enzymatically active, did not inhibit OCL formation. In addition, OIP‐2 inhibited OCL formation in cultures of highly purified human OCL precursor cells or RAW264.7 cells stimulated with 10 ng/ml of receptor activator of NF‐κB (RANK) ligand. Binding studies with His‐tagged OIP‐2 showed expression of a putative OIP‐2 receptor on RAW264.7 cells 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, rather than the mature enzyme, mediates the inhibitory effects of OIP‐2 through a putative receptor on OCL precursors.