Kennedy Institute of Rheumatology, Imperial College London
Doi
Abstract
Aggrecan is one of the most abundant components of the articular cartilage matrix and its degradation by aggrecanases is considered to be a key early event in the development of osteoarthritis. This study investigates whether the extracellular level of ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs-4), one of the most studied aggrecanases, can be regulated by receptor-mediated endocytosis. Exogenously-added ADAMTS-4 disappeared from the medium of HTB94 cells and TCA-soluble degradation products of ADAMTS-4 increased in the medium over time. Fluorescent-labeled ADAMTS-4 could be detected within the cell by confocal microscopy. ADAMTS-4 clearance by HTB94 cells and chondrocytes could be inhibited by more than 70 % by heparin and another sulfated glycan, calcium pentosan polysulfate, suggesting that the cellular uptake of ADAMTS-4 was via a mechanism involving cell surface heparan sulfate proteoglycans (HSPGs). GM6001 also inhibited ADAMTS-4 clearance by 50 %, suggesting that cell surface metalloprotease-mediated degradation played a contributory role. However, ADAMTS-4 clearance was reduced by 50 % in a HSPG-deficient cell line compared to wild-type cells, further supporting a HSPG-dependent mechanism of ADAMTS-4 cellular uptake and preliminary studies using Timp-3-null fibroblasts suggest that ADAMTS-4 may be endocytosed in a complex with TIMP-3. In HTB94 cells and chondrocytes, the appearance of TCA-soluble degradation products was also inhibited by at least 50 % by receptor-associated protein (RAP), an antagonist for members of the low-density lipoprotein receptor (LDL-R) family. However, siRNA knockdown of LDL-receptor-related protein (LRP-1), a member of the LDL-R family, in HTB94 cells and the use of an LRP-1-deficient cell line had no affect on the clearance of ADAMTS-4. These results suggest that another member of the LDL-R family may be involved. Taken together, this research has revealed a novel RAP-sensitive and/or HSPG-sensitive mechanism of ADAMTS-4 regulation which may have a role in the degradation of the cartilage matrix