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Sequence motifs of tissue inhibitor of metalloproteinases 2 (TIMP-2) determining progelatinase A (proMMP-2) binding and activation by membrane-type metalloproteinase 1 (MT1-MMP).

ABSTRACT: Fundamental cellular processes including angiogenesis and cell migration require a proteolytic cascade driven by interactions of membrane-type matrix metalloproteinase 1 (MT1-MMP) and progelatinase A (proMMP-2) that are dependent on the presence of tissue inhibitor of metalloproteinases 2 (TIMP-2). There are unique interactions between TIMP-2 and MT1-MMP, which we have previously defined, and here we identify TIMP-2 sequence motifs specific for proMMP-2 binding in the context of its activation by MT1-MMP. A TIMP-2 mutant encoding the C-terminal domain of TIMP-4 showed loss of proMMP-2 activation, indicating that the C-terminal domain of TIMP-2 is important in establishing the trimolecular complex between MT1-MMP, TIMP-2 and proMMP-2. This was confirmed by analysis of a TIMP-4 mutant encoding the C-terminal domain of TIMP-2, which formed a trimolecular complex and promoted proMMP-2 processing to the intermediate form. Mutants encoding TIMP-4 from Cys(1) to Leu(185) and partial tail sequence of TIMP-2 showed some gain of activating capability relative to TIMP-4. The identified residues were subsequently mutated in TIMP-2 (E(192)-D(193) to I(192)-Q(193)) and this inhibitor showed a significantly reduced ability to facilitate proMMP-2 processing by MT1-MMP. Furthermore, the tail-deletion mutant Delta(186-194)TIMP-2 was completely incapable of promoting proMMP-2 activation by MT1-MMP. Thus the C-terminal tail residues of TIMP-2 are important determinants for stable trimolecular complex formation between TIMP-2, proMMP-2 and MT1-MMP and play an important role in MT1-MMP-mediated processing to the intermediate and final active forms of MMP-2 at the cell surface.

SUBMITTER: Worley JR

PROVIDER: S-EPMC1223438 | biostudies-other | 2003 Jun

REPOSITORIES: biostudies-other

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Project description:Matrix metalloproteinases (MMPs) are involved in the pathology of numerous inflammatory retinal degenerations, including retinitis pigmentosa (RP). Our previous work revealed that intravitreal injections with tissue inhibitor of metalloproteinases 1 (TIMP-1) reduce the progression of rod cell death and inhibit cone cell remodeling that involves reactive gliosis in retinal Müller glial cells (MGCs) in rodent models. The underlying cellular and molecular mechanisms of how TIMP-1 functions in the retina remain to be resolved; however, MGCs are involved in structural homeostasis, neuronal cell survival and death. In the present study, MMP-9 and TIMP-1 expression patterns were investigated in a human MGC line (MIO-M1) under inflammatory cytokine (IL-1β and TNF-α) and oxidative stress (H2O2) conditions. First, both IL-1β and TNF-α, but not H2O2, have a mild in vitro pro-survival effect on MIO-M1 cells. Treatment with either cytokine results in the imbalanced secretion of MMP-9 and TIMP-1. H2O2 treatment has little effect on their secretion. The investigation of their intracellular expression led to interesting observations. MMP-9 and TIMP-1 are both expressed, not only in the cytoplasm, but also inside the nucleus. None of the treatments alters the MMP-9 intracellular distribution pattern. In contrast to MMP-9, TIMP-1 is detected as speckles. Intracellular TIMP-1 aggregation forms in the cytoplasmic area with IL-1β treatment. With H2O2 treatments, the cell morphology changes from cobbles to spindle shapes and the nuclei become larger with increases in TIMP-1 speckles in an H2O2 dose-dependent manner. Two TIMP-1 cell surface receptors, low density lipoprotein receptor-related protein-1 (LRP-1) and cluster of differentiation 82 (CD82), are expressed within the nucleus of MIO-M1 cells. Overall, these observations suggest that intracellular TIMP-1 is a target of proinflammatory and oxidative insults in the MGCs. Given the importance of the roles for MGCs in the retina, the functional implication of nuclear TIMP-1 and MMP-9 in MGCs is discussed.

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Sequence motifs of tissue inhibitor of metalloproteinases 2 (TIMP-2) determining progelatinase A (proMMP-2) binding and activation by membrane-type metalloproteinase 1 (MT1-MMP).

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