The best known function of the tissue inhibitor of matrix metalloproteinases (TIMP-1) is its regulation of metalloproteinase activity. It has been reported to inhibit tumor cell invasion in vitro and tumor growth and metastasis in vivo. Conversely, TIMP-1 is known as erythroid potentiating activity (EPA) due to its growth promoting effect on erythroid precursor cells, and it has also been shown to have growth stimulatory effect on cultured normal and neoplastic cell types. The major goal of this project was to characterize the role of TIMP-1 in endothelial cell behavior during tumor invasion and neovascularization. The first step to reach this goal was to generate recombinant TIMP-1 (rTIMP-1). A recombinant baculovirus was engineered in which human endothelial TIMP-1 cDNA was placed under the control of the polyhedrin promoter. Western blot analysis, using TIMP-1 antiserum, identified a 29 kDa protein in serum-free culture medium and a 24 kDa protein in cell lysates of the baculovirus expression system. The secreted 29 kDa protein was found to be glycosylated which has not been accomplished in other recombinant vector systems and is thought to be essential for TIMP-1 activity. A 274 base pair riboprobe was constructed which specifically hybridizes to the N-terminal domain of human endothelial TIMP-1 mRNA. This riboprobe was used for in situ hybridization studies which have so far shown tumor-cell mediated stimulation of endothelial TIMP-1 mRNA expression. This report describes for the first time the production of glycosylated rTIMP-1. The baculovirus system will serve as a high yielding source of rTIMP-1 which will be valuable for in vivo studies on the role of TIMP-1 in tumor vascular invasion and endothelial proliferation.
