The potential role of the matrix metalloproteinase (MMP) system in the pathophysiology of the adipose tissue was investigated in a mouse model of nutritionally induced obesity. mRNA levels of 16 MMPs and 4 tissue inhibitors of MMPs (TIMPs) were measured by semiquantitative RT-PCR in adipose tissue isolated from mice maintained for 15 weeks on a standard or high-fat diet. In mice on standard diet, with the exception of MMP-8, all MMP and TIMP transcripts were detected in both gonadal and subcutaneous depots. In obese mice, the expression of MMP-3, -11, -12, -13, and -14 and TIMP-1 mRNAs was upregulated, whereas that of MMP-7, -9, -16, and -24 and TIMP-4 was downregulated. Most MMP and TIMP mRNAs were expressed at higher levels in stromal-vascular cells than in mature adipocytes. Analysis of adipose tissue by in situ fluorescent zymography revealed MMP-dependent proteolytic activities, demonstrating the presence of active MMPs in the intact tissue. In vitro conversion of adipogenic 3T3-F442A cells into mature adipocytes was associated with substantial modulations of MMP and TIMP expression. Moreover, this in vitro adipogenesis was reduced in the presence of a synthetic MMP inhibitor. Thus, the adipose tissue expresses a large array of MMPs and TIMPs, which modulate adipocyte differentiation.

The increasing diversity in both substrates and functions of matrix metalloproteinases (MMPs) makes these enzymes central regulators in the complex tumor ecosystem composed of cancer cells and their microenvironment. In the majority of cancers, membrane-associated and extracellular proteases are mainly produced by host cells including inflammatory cells, endothelial cells, pericytes and fibroblasts. Recent data based on in vitro and in vivo studies have demonstrated the relevance of these enzymes in multiple processes controlling cancer growth, angiogenesis and metastatic dissemination. This review will present the emerging MMP-related features of cancer cells and host cells.

Development of vasculature and mRNA expression of 17 pro- or antiangiogenic factors were studied during adipose tissue development in nutritionally induced or genetically determined murine obesity models. Subcutaneous (SC) and gonadal (GON) fat pads were harvested from male C57Bl/6 mice kept on standard chow [standard fat diet (SFD)] or on high-fat diet for 0-15 wk and from male ob/ob mice kept on SFD. Ob/ob mice and C57Bl/6 mice on high-fat diet had significantly larger SC and GON fat pads, accompanied by significantly higher blood content, increased total blood vessel volume, and higher number of proliferating cells. mRNA and protein levels of angiopoietin (Ang)-1 were down-regulated, whereas those of thrombospondin-1 were up-regulated in developing adipose tissue in both obesity models. Ang-1 mRNA levels correlated negatively with adipose tissue weight in the early phase of nutritionally induced obesity as well as in genetically determined obesity. Placental growth factor and Ang-2 expression were increased in SC adipose tissue of ob/ob mice, and thrombospondin-2 was increased in both their SC and GON fat pads. mRNA levels of vascular endothelial growth factor (VEGF)-A isoforms VEGF-B, VEGF-C, VEGF receptor-1, -2, and -3, and neuropilin-1 were not markedly modulated by obesity. This modulation of angiogenic factors during development of adipose tissue supports their important functional role in obesity.

Membrane-type matrix metalloproteinases (MT-MMP) constitute a subfamily of six distinct membrane-associated MMPs. Although the contribution of MT1-MMP during different steps of cancer progression has been well documented, the significance of other MT-MMPs is rather unknown. We have investigated the involvement of MT4-MMP, a glycosylphosphatidylinositol-anchored protease, in breast cancer progression. Interestingly, immunohistochemical analysis shows that MT4-MMP production at protein level is strongly increased in epithelial cancer cells of human breast carcinomas compared with normal epithelial cells. Positive staining for MT4-MMP is also detected in lymph node metastases. In contrast, quantitative reverse transcription-PCR analysis reveals similar MT4-MMP mRNA levels in human breast adenocarcinomas and normal breast tissues. Stable transfection of MT4-MMP cDNA in human breast adenocarcinoma MDA-MB-231 cells does not affect in vitro cell proliferation or invasion but strongly promotes primary tumor growth and associated metastases in RAG-1 immunodeficient mice. We provide for the first time evidence that MT4-MMP overproduction accelerates in vivo tumor growth, induces enlargement of i.t. blood vessels, and is associated with increased lung metastases. These results identify MT4-MMP as a new putative target to design anticancer strategies.

Angiogenesis, the formation of new blood vessels from preexisting ones, is a key event in tumor progression controlled by a balance between positive and negative regulators (1, 2). From this observation has emerged the concept of the "angiogenic switch" in which endothelial activation status is determined by the induction of angiogenic factors and/or the loss of inhibitors (3). Positive regulators include at least vascular endothelial growth factor family (VEGF-A, -B, -C, -D), 1 fibroblast growth factors, placental-like growth factor (PlGF), angiopoietins, their tyrosine kinase receptors (VEGF-R1, -R2; Tie1 and Tie2) and neuropilin-1 (NRP-1), a co-receptor for VEGF (1, 4). An increasing number of negative regulators have been identified such as inhibitors of proteinases, thrombospondins, interferons, chemokines (IP-10 and PF-4), and bioactive fragments of the extracellular matrix (2, 4). Once a tumor has acquired an angiogenic phenotype, tumor cell migration, invasion, and vessel sprouting require the dynamic and coordinated action of many cell surface molecules, including proteinases and integrins mediating cell-matrix interactions.Among the cell-associated proteinases, matrix metalloproteinases (MMPs) anchored to the plasma membrane, called membrane-type MMPs (MT-MMPs) play a pivotal role in pericellular proteolysis. Of the six MT-MMPs that have been identified to date, four (MT1-, MT2-, MT3-, and MT5-MMP) contain a transmembrane domain followed by a cytoplasmic tail and two (MT4-and MT6-MMP) are tethered to the cell surface via a glycosylphosphatidylinositol link (for review, see . It now appears that all MT-MMPs may have the ability to activate pro-MMP2 (9 -13). Although, each of the four physiological tissue inhibitors of MMPs (TIMPs) can non selectively bind to all active MMPs, TIMP1 is unable to inhibit effectively MT1, 2, 3, and 5-MMPs (14). TIMP-2 has dual functions, inhibiting the activity of each MT-MMP and participating in the activation of pro-MMP2 through the formation of a ternary complex formed of MT1-MMP, TIMP-2, and pro-MMP2 (for review, see Refs. 7,15,and 16). MMP2 activation by MT1-MMP could be promoted by interaction with integrin (17) and oligomerization of 19).

Abstract-The effect of galardin, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, was studied in mice kept on a high fat diet (HFD). Five-week-old male wild-type mice were fed the HFD (42% fat) for up to 12 weeks and were daily injected intraperitoneally with the inhibitor (100 mg/kg) or with vehicle. After 12 weeks of the HFD, the body weights of both groups were comparable, but the weight of the isolated subcutaneous (SC) or gonadal (GON) fat deposits was significantly lower in the inhibitor-treated group than in the control group (88Ϯ11 versus 251Ϯ66

Tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for the membrane type 1 matrix metalloproteinase (MT1-MMP)-dependent activation of pro-MMP-2 on the cell surface. MT1-MMP-bound TIMP-2 has been shown to function as a receptor for secreted pro-MMP-2, resulting in the formation of a trimolecular complex. In the presence of uncomplexed active MT1-MMP, the prodomain of cell surface-associated MMP-2 is cleaved, and activated MMP-2 is released. However, the behavior of MT1-MMP-bound TIMP-2 during MMP-2 activation is currently unknown. In this study, 125 I-labeled recombinant TIMP-2 ( 125 I-rTIMP-2) was used to investigate the fate of TIMP-2 during pro-MMP-2 activation by HT1080 and transfected A2058 cells. HT1080 and A2058 cells transfected with MT1-MMP cDNA (but not vector-transfected A2058 cells) were able to bind 125 I-rTIMP-2, to activate pro-MMP-2, and to process MT1-MMP into an inactive 43-kDa form. Under these conditions, 125 IrTIMP-2 bound to the cell surface was rapidly internalized and degraded in intracellular organelles through a bafilomycin A 1 -sensitive mechanism, and 125 I-bearing low molecular mass fragment(s) were released in the culture medium. These different processes were inhibited by hydroxamic acid-based synthetic MMP inhibitors and rTIMP-2, but not by rTIMP-1 or cysteine, serine, or aspartic proteinase inhibitors. These results support the concept that the MT1-MMP-dependent internalization and degradation of TIMP-2 by some tumor cells might be involved in the regulation of pericellular proteolysis.

The close correlation observed between matrix metalloproteinase 2 (MMP-2) activation and metastatic progression in various tumors suggests that MMP-2 is a`master switch' triggering tumor spread. Recently, membrane type 1 MMP (MT1-MMP) was identified as a potential physiological activator of MMP-2. Like all other MMPs, MT1-MMP possesses a pro-domain which must be removed for the enzyme to acquire its catalytic potential. The presence of a typical recognition motif (RXKR) for the furin-like convertases at the end of its pro-domain suggests a potential role for these proteinases in MT1-MMP processing. In order to evaluate the implication of furin in pro-MT1-MMP processing, we treated HT1080 cells with a synthetic furin inhibitor and monitored their ability to activate pro-MMP-2 as well as their invasive potential. Our results demonstrated that the furin inhibitor decreased pro-MT1-MMP processing as well as pro-MMP-2 activation and cell invasiveness. Therefore, our data bring further evidence that furin is a key factor in the maturation of MMPs associated with the invasive and metastatic potential of tumor cells.z 1998 Federation of European Biochemical Societies.