Evidence for a novel insulin-like growth factor (IGF)-dependent protease regulating IGF-binding protein-4 in dermal fibroblasts.
Evidence for a novel insulin-like growth factor (IGF)-dependent protease regulating IGF-binding protein-4 in dermal fibroblasts.
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DOI：
10.1210/endo.131.5.1385096
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发表时间：
1992-11
期刊：
影响因子：
4.8
通讯作者：
J. Fowlkes;M. Freemark
中科院分区：
文献类型：
--
作者：
J. Fowlkes;M. Freemark
胰岛素样生长因子（IGF）降低细胞条件培养基中IGF结合蛋白-4（IGFBP-4）水平的机制知之甚少。IGFs对成纤维细胞条件培养基中IGFBP-4水平的影响不是通过1型或2型细胞IGF受体介导的，并且IGFs对成人成纤维细胞或大鼠神经母细胞瘤细胞中IGFBP-4信使RNA水平几乎没有影响。为了确定IGFs对IGFBP-4的作用是否可能通过改变IGFBP-4的降解而发挥，我们将来自绵羊或人皮肤成纤维细胞的无细胞成纤维细胞条件培养基与或不与IGF-I、IGF-II（各1微克/毫升）或胰岛素（10微克/毫升）在37 ℃下孵育72小时。然后通过使用放射性标记的IGF的蛋白质配体印迹和使用针对人IGFBP-4的多克隆抗血清的免疫印迹分析样品。在不存在IGFs的情况下，未观察到各种IGFBPs的基础浓度的明显变化。相比之下，与IGFs一起孵育的培养基导致绵羊和人IGFBP-4水平降低70-80%，而与胰岛素一起孵育则没有效果。类似地，含有重组人IGFBP-4的无细胞条件培养基与IGF-I的孵育引起28 K蛋白的可检测水平的降低。IGFBP-4水平的降低伴随着免疫反应性的约17- 20 K片段的出现，该片段通过配体印迹不结合放射性标记的IGF。IGF-依赖性降低IGFBP-4被阻止共孵育的介质与丝氨酸蛋白酶抑制剂，EDTA，或1，10-菲咯啉，这表明IGFs可以激活IGFBP-4特异性金属丝氨酸蛋白酶存在于成纤维细胞条件培养基。或者，IGF-I或IGF-II与IGFBP-4的结合可增强IGFBP-4对蛋白水解降解的敏感性。IGF-I和IGF-II可以直接促进IGFBP-4蛋白水解降解成不结合IGF的片段的证明提供了IGF可以增加其自身在生物流体中的可用性和/或活性的新机制。
The mechanisms by which insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) levels in cellular conditioned media are poorly understood. The effect of IGFs on IGFBP-4 levels in fibroblast conditioned media is not mediated via the type 1 or type 2 cellular IGF receptors, and the IGFs exert little or no effects on IGFBP-4 messenger RNA levels in human adult fibroblasts or in rat neuroblastoma cells. To determine whether the effects of IGFs on IGFBP-4 might be exerted through alterations in IGFBP-4 degradation, we incubated cell-free, fibroblast-conditioned media from either sheep or human dermal fibroblasts with or without IGF-I, IGF-II (each 1 microgram/ml), or insulin (10 micrograms/ml) for 72 h at 37 C. Samples were then analyzed by Western ligand blot using radiolabeled IGFs and by immunoblotting using a polyclonal antisera to human IGFBP-4. In the absence of IGFs, no apparent changes in the basal concentrations of the various IGFBPs were observed. In contrast, incubation of media with IGFs caused a 70-80% reduction in levels of both sheep and human IGFBP-4, whereas incubation with insulin was without effect. Similarly, incubation of cell-free conditioned media containing recombinant human IGFBP-4 with IGF-I caused a reduction in detectable levels of the 28K protein. The decrease in IGFBP-4 levels was accompanied by the appearance of an immunoreactive approximate 17-20K fragment that did not bind radiolabeled IGFs by ligand blot. The IGF-dependent decrease in IGFBP-4 was prevented by coincubation of the media with serine protease inhibitors, EDTA, or 1,10-phenanthrolene, suggesting that IGFs may activate an IGFBP-4 specific metallo-serine protease present in fibroblast conditioned media. Alternatively, binding of IGF-I or -II to IGFBP-4 may enhance the susceptibility of IGFBP-4 to proteolytic degradation. The demonstration that IGF-I and IGF-II can promote directly the proteolytic degradation of IGFBP-4 into fragments that do not bind IGFs provides a novel mechanism by which the IGFs may increase their own availability and/or activity in biological fluids.