Motoharu Seiki

Membrane Type 1 Matrix Metalloproteinase Digests Interstitial Collagens and Other Extracellular Matrix Macromolecules

Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed on cancer cell membranes and activates the zymogen of MMP-2 (gelatinase A). We have recently isolated MT1-MMP complexed with tissue inhibitor of metalloproteinases 2 (TIMP-2) and demonstrated that MT1-MMP exhibits gelatinolytic activity by gelatin zymography (Imai, K., Ohuchi, E., Aoki, T., Nomura, H., Fujii, Y., Sato, H., Seiki, M., and Okada, Y. (1996) Cancer Res. 56, 2707-2710). In the present study, we have further purified to homogeneity a deletion mutant of MT1-MMP lacking the transmembrane domain (ΔMT1) and native MT1-MMP secreted from a human breast carcinoma cell line (MDA-MB-231 cells) and examined their substrate specificities. Both proteinases are active, without any treatment for activation, and digest type I (guinea pig), II (bovine), and III (human) collagens into characteristic 3/4 and 1/4 fragments. The cleavage sites of type I collagen are the Gly775-Ile776 bond for α1(I) chains and the Gly775-Leu776 and Gly781-Ile782 bonds for α2(I) chains. ΔMT1 hydrolyzes type I collagen 6.5- or 4-fold more preferentially than type II or III collagen, whereas MMP-1 (tissue collagenase) digests type III collagen more efficiently than the other two collagens. Quantitative analyses of the activity of ΔMT1 and MMP-1 indicate that ΔMT1 is 5-7.1-fold less efficient at cleaving type I collagen. On the other hand, gelatinolytic activity of ΔMT1 is 8-fold higher than that of MMP-1. ΔMT1 also digests cartilage proteoglycan, fibronectin, vitronectin and laminin-1 as well as α1-proteinase inhibitor and α2-macroglobulin. The activity of ΔMT1 on type I collagen is synergistically increased with co-incubation with MMP-2. These results indicate that MT1-MMP is an extracellular matrix-degrading enzyme sharing the substrate specificity with interstitial collagenases, and suggest that MT1-MMP plays a dual role in pathophysiological digestion of extracellular matrix through direct cleavage of the substrates and activation of proMMP-2.

The Membrane-Anchored MMP Inhibitor RECK Is a Key Regulator of Extracellular Matrix Integrity and Angiogenesis

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.

MT1‐MMP: A potent modifier of pericellular microenvironment

Cells are regulated by many different means, and there is more and more evidence emerging that changes in the microenvironment greatly affect cell function. MT1‐MMP is a type I transmembrane proteinase which participates in pericellular proteolysis of extracellular matrix (ECM) macromolecules. The enzyme is cellular collagenase essential for skeletal development, cancer invasion, growth, and angiogenesis. MT1‐MMP promotes cell invasion and motility by pericellular ECM degradation, shedding of CD44 and syndecan1, and by activating ERK. Thus MT1‐MMP is one of the factors that influence the cellular microenvironment and thereby affect cell‐signaling pathways and eventually alters cellular behavior. As a proteinase, MT1‐MMP is regulated by inhibitors, but it also requires formation of a homo‐oligomer complex, localization to migration front of the cells, and internalization to become a “functionally active” cell function modifier. Developing new means to inhibit “functional activity” of MT1‐MMP may be a new direction to establish treatments for the diseases that MT1‐MMP mediates such as cancer and rheumatoid arthritis.

Induction of interleukin 2 receptor gene expression by p40x encoded by human T-cell leukemia virus type 1.

Human T‐cell leukemia virus type 1 (HTLV‐1) is an etiologic agent of adult T‐cell leukemia (ATL). A viral product, p40x, encoded by the pX sequence of HTLV‐1 is a trans‐acting transcriptional activator of the long terminal repeat (LTR) and has been suspected of involvement in leukemogenesis, activating the cellular genes. The cellular interleukin‐2 (IL‐2) and its receptor (IL‐2R), the latter of which is expressed on ATL leukemic cells, were shown to be transiently induced by transfection of plasmid pMTPX expressing pX in two T‐cell lines, Jurkat and HSB‐2, but not in other human T‐ or B‐cell lines. The cell type specificity of IL‐2R induction by pX expression was the same as that by phytohaemagglutinin/phorbol ester activation, indicating the requirement for some specific cellular factors or a certain state of cellular differentiation. Induction of IL‐2 and IL‐2R at mRNA level was also demonstrated in transfected cells. Transfections with mutants of pMTPX in which the open reading frames for p40x, p27x‐III and p21x‐III were inactivated indicated that p40x alone was sufficient for induction of the IL‐2R in inducible cells. This induction of the IL‐2R by p40x of HTLV‐1 may contribute to preferential proliferation of HTLV‐1 infected cells at an early stage of ATL development and eventually increase the number of putative target cells for malignant transformation.

Membrane-type 1 matrix metalloproteinase: a key enzyme for tumor invasion

Matrix metalloproteinases (MMPs) are believed to play a pivotal role in malignant behavior of cancer cells such as rapid tumor growth, invasion, and metastasis by degrading extracellular matrix (ECM). Different types of synthetic inhibitors against MMPs (MMPIs) were developed as candidates for anti-cancer therapeutics and so far clinical trials had led to no significant success. However, this does not diminish the importance of MMPs in the malignancy of cells. Details about MMPs, specifically when and how they take part in the development of cancer are necessary for more advanced application of MMPIs. In this paper, we summarize recent knowledge about membrane-type 1 matrix metalloproteinase (MT1-MMP) which is expressed on cancer cell surface as an invasion-promoting proteinase. By localizing at the leading edge of invasive cancer cells, MT1-MMP degrades components of the tissue barriers. One of the major targets is type I collagen, the most abundant ECM component. Although MT1-MMP itself cannot degrade type IV collagen in the basement membrane, it binds to and activates proMMP-2, one of the type IV collagenases. However, degradation of the ECM is not the sole function of MT1-MMP. MT1-MMP also regulates cell-ECM interaction by processing cell adhesion molecules such as CD44 and integrin alphav chain, and eventually promotes cell migration as well. In addition to the transcriptional regulation, invasion-promoting activity of the MT1-MMP is also strictly monitored at the post-translational level. Precise knowledge about the regulation will give us insight to develop new methods for treating invasive cancer patients.

Activation of a recombinant membrane type 1-matrix metalloproteinase (MT1-MMP) by furin and its interaction with tissue inhibitor of metalloproteinases (TIMP)-2.

Membrane type 1‐matrix metalloproteinase (MT1‐MMP) initiates the activation of the zymogen progelatinase A/72‐kDa type IV collagenase by cleavage of the Asn66‐Leu peptide bond. We previously pointed out that MT1‐MMP possesses a unique amino acid sequence Arg‐Arg‐Lys‐Arg111 which is a potential recognition sequence for furin‐like proteases (Nature, 370 (1994) 61–65). Here, using a recombinant MT1‐MMP expressed in Escherichia coli we demonstrated that furin specifically cleaves MT1‐MMP between Arg111‐Tyr in vitro, which resulted in a stimulation of progelatinase A‐activation function. Tissue inhibitor of metalloproteinases (TIMP)‐2 inhibited activation of progelatinase A by forming a stable complex with activated MT1‐MMP.

Homophilic complex formation of MT1-MMP facilitates proMMP-2 activation on the cell surface and promotes tumor cell invasion.

Activation of proMMP‐2 by MT1‐MMP is considered to be a critical event in cancer cell invasion. In the activation step, TIMP‐2 bound to MT1‐MMP on the cell surface acts as a receptor for proMMP‐2. Subsequently, adjacent TIMP‐2‐free MT1‐MMP activates the proMMP‐2 in the ternary complex. In this study, we demonstrate that MT1‐MMP forms a homophilic complex through the hemopexin‐like (PEX) domain that acts as a mechanism to keep MT1‐MMP molecules close together to facilitate proMMP‐2 activation. Deletion of the PEX domain in MT1‐MMP, or swapping the domain with the one derived from MT4‐MMP, abolished the ability to activate proMMP‐2 on the cell surface without affecting the proteolytic activities. In addition, expression of the mutant MT1‐MMP lacking the catalytic domain (MT1PEX‐F) efficiently inhibited complex formation of the full‐length enzymes and activation of pro MMP‐2. Furthermore, expression of MT1PEX‐F inhibited proMMP‐2 activation and Matrigel invasion activity of invasive human fibrosarcoma HT1080 cells. These findings elucidate a new function of the PEX domain: regulating MT1‐MMP activity on the cell surface, which accelerates cellular invasiveness in the tissue.

Membrane-Type Matrix Metalloproteinases

Matrix metalloproteinases (MMP) degrade components of extracellular matrix (ECM), and thereby regulate formation, remodeling and maintenance of tissue. Abnormal function of cell surface proteases associated with malignant tumors may contribute directly to the invasive and malignant nature of the cells. Among the MMPs associated with the tumor cell surface, gelatinase A is believed to be particularly important, since it degrades type IV collagen, and is activated in a tumor specific manner, correlating with tumor spread and poor prognosis. Activation of pro-gelatinase A is uniquely regulated by a cell-mediated mechanism. This study describes an in vitro model that mimics the cell-surface activation mechanism. The expression of MT-MMP could not be detected in normal epithelial cells, but can be seen in transformed epithelial carcinoma cells.

TIMP-2 promotes activation of progelatinase A by membrane-type 1 matrix metalloproteinase immobilized on agarose beads

Membrane-type 1 matrix metalloproteinase (MT1-MMP)/MMP-14 is the activator of progelatinase A (proGelA)/proMMP-2 on the cell surface. However, it was a paradox that a tissue inhibitor of metalloproteinase-2 (TIMP-2), which is an inhibitor of MT1-MMP, is required for proGelA activation by the cells expressing MT1-MMP. In this study, a truncated MT1-MMP having a FLAG-tag sequence at the C terminus (MT1-F) was immobilized onto agarose beads (MT1-F/B) and used to analyze the role of TIMP-2. The proteolytic activity of MT1-F/B against a synthetic peptide substrate was inhibited by TIMP-2 in a dose-dependent manner. In contrast, TIMP-2 promoted the processing of proGelA by MT1-F/B at low concentrations and inhibited it at higher concentrations. TIMP-2 promoted the binding of proGelA to the MT1-F on the beads by forming a trimolecular complex, which was followed by processing of proGelA. A stimulatory effect of TIMP-2 was observed under conditions in which unoccupied MT1-F was still available. Thus, the ternary complex is thought to act as a means to concentrate the substrate to the bead surface and to present it to the neighboring free MT1-F.

CD44 directs membrane‐type 1 matrix metalloproteinase to lamellipodia by associating with its hemopexin‐like domain

Membrane‐type 1 matrix metalloproteinase (MT1‐ MMP) localizes at the front of migrating cells and degrades the extracellular matrix barrier during cancer invasion. However, it is poorly understood how the polarized distribution of MT1‐MMP at the migration front is regulated. Here, we demonstrate that MT1‐MMP forms a complex with CD44H via the hemopexin‐like (PEX) domain. A mutant MT1‐MMP lacking the PEX domain failed to bind CD44H and did not localize at the lamellipodia. The cytoplasmic tail of CD44H, which comprises interfaces that associate with the actin cytoskeleton, was important for its localization at lamellipodia. Overexpression of a CD44H mutant lacking the cytoplasmic tail also prevented MT1‐MMP from localizing at the lamellipodia. Modulation of F‐actin with cytochalasin D revealed that both CD44H and MT1‐MMP co‐localize closely with the actin cytoskeleton, dependent on the cytoplasmic tail of CD44H. Thus, CD44H appears to act as a linker that connects MT1‐MMP to the actin cytoskeleton and to play a role in directing MT1‐MMP to the migration front. The PEX domain of MT1‐MMP was indispensable in promoting cell migration and CD44H shedding.