IGF DEPENDENT IGFBP-4 PROTEASE--CLONING AND REGULATION

胰岛素样生长因子(IGF)是两种胎儿 和出生后的生长和代谢；IGF-I是 在出生后的动物和IGF-II中生长激素活性似乎是 胎儿正常生长发育所必需的荷尔蒙。这个 血浆和其他生物体液中的IGF与特定的IGF络合 结合蛋白(IGFBPs)，其中6个已被克隆和测序。 在这些IGFBP中，IGFBP-4已被证明持续抑制 IGF-I和IGF-II以及IGFS的生物学作用必须克服 IGFBP-4的抑制作用以发挥其生物学效应 细胞水平。在最近的研究中，我和我的同事们发现 人和绵羊皮肤成纤维细胞条件培养液中一种新的蛋白酶 这会将IGFBP-4降解成不再与IGF-I或II结合的片段。 该酶的活性取决于IGF-I或IGF-II的存在， 提供了一种新的机制，通过这种机制，政府间气候变化框架可以直接增加 无细胞条件，其自身的可用性和/或活性 生物体液。这项提议的总体目标是 鉴定这种依赖于IGF的IGFBP-4酶，并检测其调控 通过激素和细胞因子，并确定其对细胞的直接影响 生长和扩散。依赖于IGF的IGFBP-4蛋白酶将是 用蛋白酶从人成纤维细胞条件培养液中纯化 抑制剂亲和层析与反相高效 液体色谱学。蛋白水解酶活性将由IGF- 重组人IGFBP-4的依赖降解。氨基酸 该酶的序列将被用来从一株 人成纤维细胞c DNA文库。编码寡核苷酸和c DNA的探针 该酶将被用来鉴定蛋白水解酶的信使核糖核酸 体外检测细胞因子和生长因子对其的调节作用。至 直接确定蛋白酶的作用，人成纤维细胞将 将含有反义构建的 可在小鼠控制下诱导的蛋白水解酶基因 金属硫烯促进剂。这样的研究应该有助于确定总体 IGF依赖的IGFBP-4蛋白在细胞增殖中的作用 和IGF行动。总而言之，这些研究应该会为 IGFS调节正常和异常的机制 成长。

Insulin-like growth factors (IGFs) are important mediators of both fetal and postnatal growth and metabolism; IGF-I is the primary mediator of growth hormone activity in the postnatal animal and IGF-II appears to be an essential hormone for normal growth and development of the fetus. The IGFs in plasma and other biological fluids are complexed to specific IGF binding proteins (IGFBPs), of which six have been cloned and sequenced. Of these IGFBPs, IGFBP-4 has been shown to consistently inhibit the biologic actions of IGF-I and II and the IGFs must overcome the inhibitory effects of IGFBP-4 in order to exert biological effects at the cellular level. In recent studies, my colleagues and I have identified in conditioned media of human and sheep skin fibroblasts a novel protease that degrades IGFBP-4 into fragments that no longer bind IGF-I or II. The activity of this protease depends upon the presence of IGF-I or II, providing a novel mechanism by which IGFs may increase directly, under cell free conditions, their own availability and/or activity in biological fluids. The overall objective of this proposal is to characterize this IGF-dependent IGFBP-4 protease, examine its regulation by hormones and cytokines, and determine its direct effects on cell growth and proliferation. The IGF-dependent IGFBP-4 protease will be purified from human fibroblast conditioned media using protease inhibitor-affinity chromatography and reverse-phase high performance liquid chromatography. Protease activity will be assessed by IGF- dependent degradation of recombinant human IGFBP-4. The amino acid sequence of the protease will be used to clone the protease cDNA from a human fibroblast cDNA library. Oligonucleotide and cDNA probes encoding the protease will be employed to characterize the protease mRNA and to examine its regulation by cytokines and growth factors in vitro. To determine directly the effects of the protease, human fibroblasts will be transfected with a plasmid containing an antisense construct of the protease cDNA which can be induced under the control of the mouse metallothionene promotor. Such studies should help determine the overall contribution of the IGF-dependent IGFBP-4 protease on cell proliferation and IGF action. Together, these studies should provide new insights into the mechanisms by which the IGFs regulate both normal and abnormal growth.