CHARACTERIZATION OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN 5-DEGRADING PROTEASES PRODUCED THROUGHOUT MURINE OSTEOBLAST DIFFERENTIATION

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is unique ly regulated throughout MC3T3-E1 osteoblast differentiation: IGFBP-5 i s first detectable in conditioned medium (CM) of replicating preosteob lasts (day 5); IGFBP-5 levels peak between culture days 8-12, then dec line to almost undetectable levels in mature osteoblast cultures (>day 18) despite the persistence of IGFBP-5 messenger RNA. These observati ons suggest that IGFBP-5 concentrations may be regulated by posttransl ational mechanisms. To determine whether proteolysis contributes to th e disappearance of IGFBP-5 in CM of mature osteoblasts, serial samples of MC3T3-E1 cell CM obtained during a 30-day culture period were anal yzed for IGFBP-5-degrading protease activity. Using [I-125]recombinant human IGFBP-5 substrate zymography, we demonstrated that proteases wi th M(r) of 52-72 and 97 kilodaltons (kDa) were present in CM, and prot ease activity increased in concentration as cultures matured. The 52- to 72-kDa proteases were cation dependent and were inhibited by tissue inhibitor of metalloproteinase 1, a specific inhibitor of matrix meta lloproteinases (MMPs), identifying them as MMPs. Furthermore, antisera to human MMP-1 and -2 immunoprecipitated IGFBP-5-degrading proteases with M(r) of 52 and 69/72 kDa, respectively, suggesting that homologou s murine MMPs degrade IGFBP-5. Finally, MC3T3-E1 cell CM contained imm unoreactive MMP-1 and -2, and MMP-2, in particular, increased signific antly throughout differentiation. In contrast, the 97-kDa protease was neither inhibited by tissue inhibitor of metalloproteinase 1 nor immu noprecipitated by antisera to MMPs, suggesting that the 97-kDa proteas e is not a MMP. Together, these data suggest that MMPs along with an u nidentified 97-kDa protease degrade IGFBP-5 in MC3T3-E1 cell cultures. Because truncated forms of IGFBP-5 have been shown to enhance the act ion of IGF in bone cells, IGFBP-5 proteases may be instrumental in IGF -mediated bone morphogenesis.