Tissue Inhibitors of Metalloproteinases

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There are four known endogenous inhibitors of MMPs that have been cloned to date: TIMP-1, -2, -3, and -4. These four members of the TIMP family have been shown to inhibit active MMPs equally well, although some limited degree of specificity exists. For example, TIMP-2 binds preferentially to MMP-2, whereas TIMP-1 binds to MMP-9 with higher affinity than to other MMPs. The amino acid sequence of TIMPs is highly conserved (~45%) and includes 12 cysteine residues that form 6 disulfide-bonded loop structures. The junction of the first three loops has been identified as the MMP inhibitory site by both biochemical and structural studies. In fact, the first three loops expressed alone as a truncated protein have been shown to be sufficient to inhibit MMP activity. These three loops have been popularly referred to as the N-terminal domain. The C-terminal domain, comprising the remaining three disulfide-bonded loops, is more variable among the TIMP family members in terms of its primary structure and is believed to confer some of the specificity observed for certain TIMPs [5]. For example, interactions between the C-terminal domain of TIMP-2 and the PEX domain of MMP-2 are thought to stabilize the enzyme inhibitor complex, as well as to promote pro-MMP-2 activation in a trimeric complex with MT1-MMP.

It is becoming increasingly clear that TIMPs are multifunctional proteins that affect a variety of cellular activities, such as cell growth and apoptosis. Most prominent among these functions is the ability of certain TIMPs to regulate vascular and tumor cell growth. For many years, this potential has been predicated on their ability to directly inhibit the MMP activity by binding to the enzymes' active site. Since the first report that TIMPs could inhibit angiogenesis in vitro and in vivo [6], it has largely been assumed that all TIMPs inhibit angiogenesis. However, recent studies now illustrate important differences in the ability of these family members to be bona fide inhibitors of angiogenesis in vivo. Although TIMP-1, TIMP-2, and TIMP-3 are all inhibitors of capillary EC migration, only TIMP-2 has been shown to inhibit the proliferation of normal capillary endothelial cells [7]. TIMP-1 has actually been found to be a modest stimulator of capillary EC proliferation as well as angiogenesis in vivo, whereas TIMP-3 has no significant effect on normal capillary EC proliferation. TIMP-4 has not yet been tested in these systems.

These results led us to hypothesize that the antiprolifera-tive effects of TIMP-2 are, in fact, a unique feature of this TIMP and represent a second antiangiogenic activity in this molecule. Recently, it has been reported that this second antiangiogenic site which is responsible for directly inhibiting capillary EC proliferation, is housed in the C-terminal domain of TIMP-2 and, more specifically, in Loop 6. (Figure 2) Interestingly, while the antiproliferative domain of TIMP-2 was a potent inhibitor of corneal neovasculariza-tion when angiogenesis was stimulated by the exogenous addition of an angiogenic mitogen, the MMP-inhibitory domain resulted in only modest inhibition that was not statistically different from a mutant form of the protein that lacked MMP inhibitory activity [8]. In fact, synthetic MMP inhibitors, such as BB-94, do not inhibit angiogenesis in this system, nor do monospecific, MMP-2 immuno-

TIMP-2

T2N

T2C

Loop 6

• Inhibits MMP activity

• Inhibits MMP

» Inhibits capillary

' Inhibits capillary

- Inhibits capillary EC

activity

EC proliferation

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proliferation

- Inhibits embryonic

* Inhibits embryonic

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• Inhibits embryonic

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angiogenesis in

angiogenesis in the

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the CAM

the CAM

CAM

* Inhibits growth

* Inhibits growth

• Inhibits growth

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factor-stimulatec

factor-stimulated

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angiogenesis in

angiogenesis in the

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the mouse

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corneal pocket

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Figure 2 Uncoupling of the anti-angiogenic domains of TIMP-2. TIMP-2 possesses two structurally independent anti-angiogenic domains, T2N and T2C. T2N inhibits MMP activity but not capillary endothelial cell proliferation, while T2C inhibits capillary endothelial cell proliferation but not MMP activity. T2N inhibits embryonic angiogenesis in vivo as demonstrated in the chick chorioallantoic membrane (CAM) assay, while T2C inhibits both embryonic and mitogen-stimulated angiogenesis, as demonstrated in the CAM and in the mouse corneal pocket assays. The antiproliferative activity of T2C has been further isolated to Loop 6 of TIMP-2. This 24 amino acid domain is, in and of itself, a potent inhibitor of angiogenesis in vivo as shown in the two distinct in vivo models.

function plays a key role in determining their ultimate effect in vivo both with respect to regulating the vasculature as well as other important physiological systems.

Glossary

Angiogenesis: The process of new capillary formation from a preexisting vessel.

Matrix metalloproteinases (MMPs): A multigene family of metal-dependent enzymes, whose activity is considered to be a rate-limiting step of extracellular matrix degradation. The growing family of MMPs is currently composed of 28 members.

Tissue inhibitors of metalloproteinases (TIMPs): A family of endogenous inhibitors of metalloproteinases. Four family members have been cloned and expressed to date.

Acknowledgments

The authors are grateful for the support of NIH-2PO1CA455, NIH P50DK065298 and The Fortin Charitable Foundation. The authors also acknowledge Kristin Gullage for figure preparation.

References neutralizing antibodies. These results suggest that direct MMP inhibition alone may not be sufficient to inhibit the robust angiogenesis associated with pathological neovascu-larization[8].

It is possible th.at the antiproliferative effects of TIMP-2 are the result of its interaction with the PEX domain of MMP-2. As mentioned previously, TIMP-2 has been shown to participate in the cell surface activation of pro-MMP-2, and the C-terminal domain alone has been shown to inhibit this process, presumably by hijacking pro-MMP-2 from the activating complex. This interaction has now been shown to be mediated by the C-terminal tail of TIMP-2, in that a mutant form of the TIMP-2 carboxy-terminal domain containing the C-terminal tail of TIMP-4 did not result in the inhibition of pro-MMP-2 activation [9]. Given these results, it is unlikely that the antiangiogenic effects of Loop 6, which is not involved in these interactions, is the result of indirect MMP inhibition.

Much remains to be learned about the multifunctional and sometimes contradictory activities of TIMPs. For example, TIMP overexpression in cancer cell lines has been shown to result in decreased tumor growth and tumor cell colonization in some systems, while stimulating tumor growth in other systems. Similarly, whereas certain TIMPs inhibit apoptosis, others have been shown to promote it. These effects often vary depending on the specific TIMP and cell type tested and the method of gene delivery utilized. These studies continue to teach us that the physiological context in which these inhibitors and their cognate enzymes

1. Visse, R., and Nagase, H. (2003). Matrix metalloproteinases and tissue inhibitors of metalloproteinases: Structure, function, and biochemistry.

Circ. Res. 92, 827-839. A recent and comprehensive review of MMPs and TIMPs.

2. Fang, J., Shing, Y., Wiederschain, D., Yan, L., Butterfield, C., Jackson, G., Harper, J., Tamvakopoulos, G., and Moses, M. A. (2000). Matrix metalloproteinase-2 is required for the switch to the angiogenic pheno-type in a tumor model. Proc. Natl. Acad. Sci. USA 97, 3884-3889.

3. Bergers, G., Brekken, R., McMahon, G., Vu, T. H., Itoh, T., Tamaki, K., Tanzawa, K., Thorpe, P., Itohara, S., Werb, Z., and Hanahan, D. (2000). Matrix metalloproteinase-9 triggers the angiogenic switch during car-cinogenesis. Nat. Cell Biol. 2, 737-744.

4. Moses, M. A., Wiederschain, D., Loughlin, K. R., Zurakowski, D., Lamb, C. C., and Freeman, M. R. (1998). Increased incidence of matrix metalloproteinases in urine of cancer patients. Cancer Res. 58, 1395-1399.

5. Brew, K., Dinakarpandian, D., and Nagase, H. (2000). Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim. Bio-phys. Acta 1477, 267-283.

6. Moses, M. A., Sudhalter, J., and Langer, R. (1990). Identification of an inhibitor of neovascularization from cartilage. Science 248, 1408-1410.

7. Murphy, A. N., Unsworth, E. J., and Stetler-Stevenson, W. G. (1993). Tissue inhibitor of metalloproteinases-2 inhibits bFGF-induced human microvascular endothelial cell proliferation. J. Cell Physiol. 157, 351-358.

8. Fernandez, C. A., Butterfield, C., Jackson, G., and Moses, M. A. (2003). Structural and functional uncoupling of the enzymatic and angiogenic inhibitory activities of tissue inhibitor of metalloproteinase-2 (TIMP-2): loop 6 is a novel angiogenesis inhibitor. J. Biol. Chem. 278, 40989-40995. A recent structure/function analysis that identifies and characterizes the antiangiogenic domains of TIMP-2.

9. Kai, H. S., Butler, G. S., Morrison, C. J., King, A. E., Pelman, G. R., and Overall, C. M. (2002). Utilization of a novel recombinant myoglobin fusion protein expression system to characterize the TIMP-4 and TIMP-2 C-terminal domain and tails by mutagenesis: The importance of acidic residues in binding the MMP-2 Hemopexin C domain. J. Biol. Chem. 8, 8.

Capsule Biography

The authors are research scientists in the Vascular Biology Program at Children's Hospital and Harvard Medical School. Dr. Moses is an Associate Professor in the Department of Surgery at Harvard Medical School. Work from this group demonstrated for the first time that the inhibition of

MMP activity resulted in the inhibition of angiogenesis and subsequent tumor growth, and that MMP activity was critical for the switch to the angiogenic phenotype. They have published extensively on the role of MMPs and TIMPs in the regulation of vascular growth and angiogenesis.

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