Search

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an endogeneous inhibitor of metalloproteinases (MMPs), adamalysins (ADAMs) and adamalysins with thrombospondin motifs (ADAMTSs), and consequently TIMP-3 is an important regulator of extracellular matrix (ECM) turnover. It has been shown that TIMP-3 levels in the tissue are regulated post-translationally by endocytosis. This study aimed to characterize the mechanism of TIMP-3 endocytosis in more detail. In this thesis, I demonstrated that TIMP-3 is endocytosed and degraded by a number of cell types including chondrocytes, fibroblasts, and monocytes. I found that the endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1) plays a major role in TIMP-3 endocytosis. Nevertheless, I found that TIMP-3 can also be internalized by LRP-1-null cells, indicating that an LRP-1-indepentent endocytic pathway also occurs. CHO-cells lacking heparin sulphate proteoglycans (HSPGs) efficiently endocytosed TIMP-3, indicating that HSPGs are not involved in its endocytosis. Shedding of LRP-1 has been shown to reduce cell surface levels of the receptor, and hence to modulate endocytosis of its ligands. I demonstrated that LRP-1 shedding leads to accumulation of soluble LRP-1 (sLRP-1) in the medium of chondrosarcoma cells. sLRP-1 interacted with TIMP-3 and inhibited its endocytosis, but had no effect on TIMP-3 inhibition of target metalloproteinases, indicating that sLRP-1 negatively regulates TIMP-3 endocytosis. TIMP-3 endocytosis was not affected by addition of GM6001, which inhibits interaction with target metalloproteinases, indicating that TIMP-3 can be internalized independently. Internalization of TIMP-3 in complex with a number of metalloproteinases occurred with the same kinetics as that of TIMP-3 alone. Furthermore, TIMP-3 promoted the endocytosis of MMP-1, suggesting that TIMP-3 can mediate the scavenging of metalloproteinases by cells. The study thus identifies LRP-1 as a central mediator of TIMP-3 endocytosis, and indicates that this process regulates levels of TIMP-3 in the ECM, thus contributing to the maintenance of tissue homeostasis.