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

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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.