IGF-DEPENDENT IGFBP-4 PROTEASE--CLONING AND REGULATION

胰岛素样生长因子（IGF）是胎儿生长和发育的重要介质 和出生后的生长和代谢; IGF-I是主要的介质， 出生后动物的生长激素活性和IGF-II似乎 胎儿正常生长发育所必需的激素。 的 血浆和其他生物体液中的IGF与特定的IGF复合 结合蛋白（IGFBPs），其中六个已被克隆和测序。 在这些IGFBP中，IGFBP-4已经显示出持续抑制IGFBP-1的表达。 IGF-I和IGF-II的生物学作用，IGFs必须克服 IGFBP-4的抑制作用，以在细胞中发挥生物学作用。 细胞水平。 在最近的研究中，我和我的同事发现 在人和绵羊皮肤成纤维细胞的条件培养基中， 将IGFBP-4降解成不再结合IGF-I或II的片段。 这种蛋白酶的活性取决于IGF-1或II的存在， 提供了一种新的机制，IGFs可以直接增加， 无细胞条件、它们自身的可用性和/或活性， 生物液体 本建议的总体目标是 鉴定这种IGF依赖性IGFBP-4蛋白酶， 通过激素和细胞因子，并确定其对细胞的直接影响， 生长和增殖。 IGF-依赖性IGFBP-4蛋白酶将被 使用蛋白酶从人成纤维细胞条件培养基中纯化 亲和层析和反相高效液相色谱 液相色谱法 蛋白酶活性将通过IGF-ELISA进行评估。 重组人IGFBP-4的依赖性降解。 的氨基酸 蛋白酶的序列将用于从一个 人成纤维细胞cDNA文库。 寡核苷酸和cDNA探针编码 蛋白酶将用于表征蛋白酶mRNA， 在体外检测细胞因子和生长因子对其的调节。 到 直接决定蛋白酶的作用，人成纤维细胞将 用含有反义构建体的质粒转染 蛋白酶cDNA，其可以在小鼠的控制下被诱导 金属硫酮促进剂。 这些研究有助于确定总体的 IGF依赖性IGFBP-4蛋白酶对细胞增殖的贡献 IGF行动 总之，这些研究应该提供新的见解， IGFs调节正常和异常的机制 增长

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.