Tissue Inhibitor of Metalloproteinase-4 in Human Breast Cancer and Endothelial Cells

摘要

Angiogenesis is a fundamental process by which new capillaries are formed from preexisting blood vessels and it plays a very important role in both normal and pathological processes such as coronary collateral circulation, cancer growth, invasion, and metastasis, and rheumatic diseases. Angiogenesis requires breakdown of the extracellular matrix (ECM) by proteinases. Angiogenic switching is accompanied by up-regulation of matrix metalloproteinase (MMP) bioactivity and down-regulation of tissue inhibitors of metalloproteinases (TIMPs). The TIMP family may regulate ECM turnover and tissue remodeling by forming tight binding inhibitory complexes with MMPs. Recently, tissue inhibitor of metalloproteinase-4 (TIMP-4) has been reported. TIMP-4 may inhibit breast cancer cell invasion and angiogenesis. The kinetic studies of the inhibition of the MMPs by recombinant TIMP-4 protein (rTIMP-4p) was performed in a continuous fluorimetric assay with a quenched fluorescent peptide substrate. The inhibition kinetics of rTIMP-4p were analyzed against human fibroblast collagenase (MMP-1), human fibroblast gelatinase A (MMP-2), human fibroblast stromelysin (MMP-3), matrilysin (MMP-7), and human neutrophil gelatinase B (MMP-9). The inhibitor concentrations that inhibited 50% of MMP activities (IC50) were determined to be 19, 3, 45, 8, and 83 nM for MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, respectively. Therefore, TIMP-4 is a potent inhibitor of all five MMPs tested and it has preference for MMP-2 and MMP-7. We have also prepared polyclonal and specific antibody directed against an unique peptide sequence of TIMP-4. This antibody has been characterized in TIMP-4 transfected human breast cancer cell line, MDA-MB-435. Our preliminary studies have also demonstrated that human endothelial cells (HUV-EC-C and ECV304) produce pro-MMP2 pro-MMP9, and MT1-MMP, as well as TIMP-4. The expression and function of TIMP-4 during angiogenesis are under investigation.