TIMP-3 Binds to Sulfated Glycosaminoglycans of the Extracellular Matrix

Abstract

Of the four known tissue inhibitors of metalloproteinases (TIMPs), TIMP-3 is distinguished by its tighter binding to the extracellular matrix. The present results show that glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfates A, B, and C, and sulfated compounds such as suramin and pentosan efficiently extract TIMP-3 from the postpartum rat uterus. Enzymatic treatment by heparinase III or chondroitinase ABC also releases TIMP-3, but neither one alone gives complete release. Confocal microscopy shows colocalization of heparan sulfate and TIMP-3 in the endometrium subjacent to the lumen of the uterus. Immunostaining of TIMP-3 is lost upon digestion of tissue sections with heparinase III and chondroitinase ABC. The N-terminal domain of human TIMP-3 was expressed and found to bind to heparin with affinity similar to that of full-length mouse TIMP-3. The A and B ␤-strands of the N-terminal domain of TIMP-3 contain two potential heparin-binding sequences rich in lysine and arginine; these strands should form a double track on the outer surface of TIMP-3. Synthetic peptides corresponding to segments of these two strands compete for heparin in the DNase II binding assay. TIMP-3 binding may be important for the cellular regulation of activity of the matrix metalloproteinases.The extracellular matrix (ECM) 1 provides mechanical support to cells and regulates signals reaching the cell that govern cell localization, differentiation, proliferation, and apoptosis. Components of the ECM, particularly the glycosaminoglycans (GAGs), are able to sequester bioactive molecules such as growth factors (1), proteases (2), and inhibitors. Turnover of the ECM is a highly regulated process necessary for movement of cells and for release of growth factors. Matrix metalloproteases (MMPs) are believed to be key participants in this remodeling; there are at least 20 MMPs, all able to digest various ECM components (3, 4).The MMPs, in turn, are regulated by tissue inhibitors of metalloproteinases or TIMPs. The major function of the TIMPs is to inhibit MMPs; any imbalance in which the activities of MMPs outweigh the TIMP levels will favor tissue destruction and pathological processes (5, 6). The TIMPs also possess growth stimulatory and regulatory activities (7,8). The four members of the TIMP family all have similar secondary structures of six loops stabilized by six highly conserved disulfide bonds. The TIMPs all bind tightly, albeit with widely varying affinity, to the various MMPs. The x-ray structure (9) shows that the N-terminal cysteine chelates the active site zinc. TIMPs have N-and C-terminal domains, each with three loops. The N-terminal domain of TIMP-1 folds readily and displays full inhibitory activity (10).TIMP-3 has several features that distinguish it from the other TIMPs. First, it is the only TIMP to bind tightly to the ECM: it was first observed as a transformation-sensitive protein bound to the ECM of chick embryo fibroblasts (11) and extractable with SDS or guanidine. This protein was subsequently shown ...

Enhanced metastatic dissemination to multiple organs by melanoma and lymphoma cells in timp-3−/− mice

“…Our study provides the first direct evidence for an inhibitory role of host stromal timp-3 À/À during metastatic colonization. TIMPs are secreted proteins, and TIMP-3 is uniquely ECM-bound (Yu et al, 2000). Decreased TIMP-3 expression in the stroma adjacent to highly aggressive colorectal adenocarcinomas is thought to allow increased local invasion (Powe et al, 1997).…”

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3

“…However, the finding that overexpression of TIMP-3 resulted in a significantly stronger reduction of invasion stresses the importance of the properties of TIMP-3 that are distinct from those of TIMP-1. The ability to inhibit MT-MMPs and the association to the extracellular matrix 22 may explain the stronger effects of TIMP-3. The abundant presence of MT-MMPs at the invasive front of the synovial tissue in RA patients compared with the synovial tissues of patients with osteoarthritis or without arthritis 7,23,10 supports the view that MT-MMPs play an important role in cartilage destruction in RA.…”

ADAMTS-1 and -4 are up-regulated following transient middle cerebral artery occlusion in the rat and their expression is modulated by TNF in cultured astrocytes

“…ADAMTS-1, -4, -5 and -9 have aggrecanase activity and are implicated in cartilage degradation [8], [40], [43]. The ADAMTS proteoglycanases are inhibited by tissue inhibitor of metalloproteinases (TIMP)-3 which, like the ADAMTSs, is sequestered in the ECM via interactions with sulphated glycosaminoglycans [11], [18], [47]. ADAMTS expression has been detected in the CNS [1], [17], [37] and is known to be altered in disease states [10], [22], [23].…”