Thomas Crabbe

The TIMP2 Membrane Type 1 Metalloproteinase “Receptor” Regulates the Concentration and Efficient Activation of Progelatinase A A KINETIC STUDY

We have used C-terminal domain mutants to further define the role of interactions of progelatinase A and membrane type 1 matrix metalloproteinase (MT1 MMP) in the binding of TIMP2 and in the cell-associated activation of progelatinase A. Soluble constructs of MT1 MMP were used to demonstrate that binding with TIMP2 occurs primarily through N-terminal domain interactions, leaving the C-terminal domain free for interactions with progelatinase A. The rate of autolytic activation of progelatinase A initiated by MT1 MMP cleavage could be potentiated by concentration of the proenzyme by binding to heparin. Residues 568–631 of the progelatinase A C-terminal domain are important in formation of the heparin binding site, since replacement of this region with the corresponding stromelysin-1 sequence abolished binding to heparin and the potentiation of activation. The same region of gelatinase A was required for binding of latent and active enzyme to TIMP2, but residues 418–474 were not important. A similar pattern was seen using cell membrane-associated MT1 MMP; residues 568–631 were required for binding and activation of progelatinase A, whereas residues 418–474 were not. Neither region was required for activation in solution. The addition of TIMP2 to HT1080 membrane preparations expressing MT1 MMP, but depleted of endogenous TIMP2, resulted in potentiation of progelatinase A activation. This effect was dependent upon TIMP2 binding to MT1 MMP rather than at an independent membrane site. Together, the data suggest that TIMP2 forms a receptor with MT1 MMP that regulates the concentration and efficient generation of functionally active gelatinase A.

Regulation of Matrix Metalloproteinase Activitya

Matrix metalloproteinases (MMPs) are thought to initiate the degradation of the extracellular matrix during the remodeling of connective tissues. A clear understanding of the mechanisms governing the regulation of their activity during normal physiological processes should give further insights into the uncontrolled remodeling occurring in degradative pathologies. Regulation of the MMPs occurs at the level of gene expression, with precise spatial and temporal compartmentalization of both synthesis and secretion by resident cells as well as by those cells invading the tissue. Extracellularly, MMPs are further regulated by the extent of processing of the proform to an active enzyme and by the relative production of the specific inhibitors of MMPs, the tissue inhibitors of metalloproteinases (TIMPs). Furthermore, the potential for association of MMPs with the cell surface or extracellular matrix components further constrains their relationship with substrates, activators and inhibitors, acting as a further regulator of MMP activity. We initiated a program of study of the MMPs and TIMPs to ascertain the relation between their structure and their function, with particular emphasis on the mechanisms of biological regulation. Recombinant wild-type proteins and specific mutants, including deletion and site mutations, have been prepared using a mammalian expression system.’ Aspects of our findings, which illustrate the fundamental importance of the domain structure of MMPs and TIMPs in their biology, are presented here.

The C-terminal (haemopexin-like) domain structure of human gelatinase A (MMP2): structural implications for its function.

In common with most other matrix metalloproteinases, gelatinase A has a non‐catalytic C‐terminal domain that displays sequence homology to haemopexin. Crystals of this domain were used by molecular replacement to solve its molecular structure at 2.6 Å resolution, which was refined to an R value of 17.9%. This structure has a disc‐like shape, with the chain folded into a β‐propeller structure that has pseudo four‐fold symmetry. Although the topology and the side‐chain arrangement are very similar to the equivalent domain of fibroblast collagenase, significant differences in surface charge and contouring are observable on 1 side of the gelatinase A disc. This difference might be a factor in allowing the gelatinase A C‐terminal domain to bind to natural inhibitor TIMP‐2.

Human progelatinase A can be activated by matrilysin

The activation of human progelatinase A by other matrix metalloproteinases was studied by following both the loss of its N‐terminal propeptide and the accompanying increase in the rate of hydrolysis of a synthetic substrate. Activated stromelysin 1 was unable to cause any activation of progelatinase A beyond that slowly occuring by autolysis, but an 8 h incubation with activated matrilysin was able to produce 64% of the activity generated by incubation with (4‐aminophenylmercuric)acetate (APMA). Wild‐type progelatinase A and a mutant proenzyme that cannot become active were both cleaved by matrilysin to a lower molecular weight species that had lost the propeptide. This shows that matrilysin activates progelatinase A by removing the propeptide in a process that does not require any autolytic cleavages.

Dissecting the Role of Matrix Metalloproteinases (MMP) and Integrin αvβ3 in Angiogenesis In vitro: Absence of Hemopexin C Domain Bioactivity, but Membrane-Type 1-MMP and αvβ3 Are Critical

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin αvβ3–mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti–membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin αvβ3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin αvβ3. Moreover, no specific binding of pro-MMP-2 to integrin αvβ3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin αvβ3, and this in a PEX-independent manner. Likewise, integrin αvβ3–expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and αvβ3- dependent but MMP-2 independent and does not support a role for PEX in αvβ3 integrin binding or in modulating angiogenesis in this system.