Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor

Abstract

The tissue inhibitor of metalloproteinases-3 (TIMP-3) is a major regulator of extracellular matrix turnover and protein shedding by inhibiting different classes of metalloproteinases, including disintegrin metalloproteinases (ADAMs). Tissue bioavailability of TIMP-3 is regulated by the endocytic receptor low-density-lipoprotein receptor-related protein-1 (LRP-1). TIMP-3 plays protective roles in disease. Thus, different approaches have been developed aiming to increase TIMP-3 bioavailability, yet overall effects of increased TIMP-3 in vivo have not been investigated. Herein, by using unbiased mass-spectrometry we demonstrate that TIMP-3-overexpression in HEK293 cells has a dual effect on shedding of transmembrane proteins and turnover of soluble proteins. Several membrane proteins showing reduced shedding are known as ADAM10 substrates, suggesting that exogenous TIMP-3 preferentially inhibits ADAM10 in HEK293 cells. Additionally identified shed membrane proteins may be novel ADAM10 substrate candidates. TIMP-3-overexpression also increased extracellular levels of several soluble proteins, including TIMP-1, MIF and SPARC. Levels of these proteins similarly increased upon LRP-1 inactivation, suggesting that TIMP-3 increases soluble protein levels by competing for their binding to LRP-1 and their subsequent internalization. In conclusion, our study reveals that increased levels of TIMP-3 induce substantial modifications in the cellular secretome and that TIMP-3-based therapies may potentially provoke undesired, dysregulated functions of ADAM10 and LRP-1.

TIMP-3 facilitates binding of target metalloproteinases to the endocytic receptor LRP-1 and promotes scavenging of MMP-1

“…In addition, the catalytic activity of several metalloproteinases can be post-translationally activated by proteolytic removal of their inhibitory pro-domain 4,6 . Moreover, a major mechanism that regulates the extracellular levels of several MMPs (including MMP-2 8 , MMP-9 9 and MMP-13 10,11 ) ADAMTSs (including ADAMTS-1 12 , ADAMTS-4 13 and ADAMTS-5 14 ) and TIMPs (including TIMP-1 15 , TIMP-2 8 , TIMP-3 16,17 ) is endocytosis and subsequent lysosomal degradation via the scavenger receptor low-density lipoprotein receptor-related protein 1 (LRP-1) 8,9 . LRP-1 is considered to be a master regulator of ECM turnover, since the affinity of individual metalloproteinases and TIMPs for LRP-1 determines their extracellular half-life and so controls the balance between ECM degradation and deposition.…”

Targeting ADAM10 in Cancer and Autoimmunity

“…Another recent study using HEK293 cells that overexpress TIMP-3 show evidence of reduced ADAM10-specific substrates after secretome analysis by mass spectrometry label free quantification. This study showed that with TIMP-3 overexpression, several ligands for the low-densitylipoprotein receptor-related protein-1 (LRP-1) are upregulated, such as macrophage inhibitor factor (MIF) (88). As TIMP-3 also binds to LRP-1, overexpression of TIMP-3 outcompetes other ligands of LRP-1 and thus the cell compensates by increasing expression (88).…”

“…This study showed that with TIMP-3 overexpression, several ligands for the low-densitylipoprotein receptor-related protein-1 (LRP-1) are upregulated, such as macrophage inhibitor factor (MIF) (88). As TIMP-3 also binds to LRP-1, overexpression of TIMP-3 outcompetes other ligands of LRP-1 and thus the cell compensates by increasing expression (88). The authors of this study warn that the use of TIMP-3 interventions as an ADAM10 regulator could yield these alterations to the secretome and results might be unanticipated.…”

Role of ADAM10 as a CD30 Sheddase in Classical Hodgkin Lymphoma

“…Particularly, fibroblasts of the nodular sclerosing subtype of cHL express high amounts of the tissue inhibitor of metalloproteinases 3 (TIMP3) (18). Although TIMP3 principally inhibits ADAM10 and ADAM17, it predominantly blocks ADAM10 in vivo (19). Thus, sheddase inhibition in the tumor microenvironment of certain cases of cHL might influence the amount of EVassociated CD30.…”