Thrailkill K M, Quarles L D, Nagase H, Suzuki K, Serra D M, Fowlkes J L
Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Endocrinology. 1995 Aug;136(8):3527-33. doi: 10.1210/endo.136.8.7543045.
Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is uniquely regulated throughout MC3T3-E1 osteoblast differentiation: IGFBP-5 is first detectable in conditioned medium (CM) of replicating preosteoblasts (day 5); IGFBP-5 levels peak between culture days 8-12, then decline to almost undetectable levels in mature osteoblast cultures (> day 18) despite the persistence of IGFBP-5 messenger RNA. These observations suggest that IGFBP-5 concentrations may be regulated by posttranslational mechanisms. To determine whether proteolysis contributes to the disappearance of IGFBP-5 in CM of mature osteoblasts, serial samples of MC3T3-E1 cell CM obtained during a 30-day culture period were analyzed for IGFBP-5-degrading protease activity. Using [125I]recombinant human IGFBP-5 substrate zymography, we demonstrated that proteases with M(r) of 52-72 and 97 kilodaltons (kDa) were present in CM, and protease 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 metalloproteinases (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 homologous murine MMPs degrade IGFBP-5. Finally, MC3T3-E1 cell CM contained immunoreactive MMP-1 and -2, and MMP-2, in particular, increased significantly throughout differentiation. In contrast, the 97-kDa protease was neither inhibited by tissue inhibitor of metalloproteinase 1 nor immunoprecipitated by antisera to MMPs, suggesting that the 97-kDa protease is not a MMP. Together, these data suggest that MMPs along with an unidentified 97-kDa protease degrade IGFBP-5 in MC3T3-E1 cell cultures. Because truncated forms of IGFBP-5 have been shown to enhance the action of IGF in bone cells, IGFBP-5 proteases may be instrumental in IGF-mediated bone morphogenesis.
胰岛素样生长因子（IGF）结合蛋白-5（IGFBP-5）在MC3T3-E1成骨细胞分化过程中受到独特调控：在增殖的前成骨细胞（第5天）的条件培养基（CM）中首次可检测到IGFBP-5；IGFBP-5水平在培养第8 - 12天达到峰值，然后在成熟成骨细胞培养物（>第18天）中降至几乎不可检测的水平，尽管IGFBP-5信使核糖核酸持续存在。这些观察结果表明，IGFBP-5浓度可能受翻译后机制调控。为了确定蛋白水解是否导致成熟成骨细胞CM中IGFBP-5的消失，对在30天培养期内获得的MC3T3-E1细胞CM的系列样本进行了IGFBP-5降解蛋白酶活性分析。使用[125I]重组人IGFBP-5底物酶谱法，我们证明CM中存在分子量为52 - 72和97千道尔顿（kDa）的蛋白酶，并且随着培养物成熟，蛋白酶活性浓度增加。52至72 kDa的蛋白酶依赖阳离子，并且被基质金属蛋白酶（MMP）的特异性抑制剂金属蛋白酶组织抑制剂1抑制，将它们鉴定为MMP。此外，针对人MMP-1和-2的抗血清分别免疫沉淀了分子量为52和69/72 kDa的IGFBP-5降解蛋白酶，表明同源鼠MMP降解IGFBP-5。最后，MC3T3-E1细胞CM含有免疫反应性MMP-1和-2，尤其是MMP-2在整个分化过程中显著增加。相比之下，97 kDa的蛋白酶既不被金属蛋白酶组织抑制剂1抑制，也不被针对MMP的抗血清免疫沉淀，表明97 kDa的蛋白酶不是MMP。总之，这些数据表明MMP与一种未鉴定的97 kDa蛋白酶在MC3T3-E1细胞培养物中降解IGFBP-5。由于已证明IGFBP-5的截短形式可增强IGF在骨细胞中的作用，IGFBP-5蛋白酶可能在IGF介导的骨形态发生中起作用。
