Expression and tissue localization of membrane-type 1, 2, and 3 matrix metalloproteinases in human astrocytic tumors.
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Three different membrane-type matrix metalloproteinases (MT1-, MT2-, and MT3-MMPs) are known to activate in vitro the zymogen of MMP-2 (pro-MMP-2, progelatinase A), which is one of the key MMPs in invasion and metastasis of various cancers. In the present study, we have examined production and activation of pro-MMP-2, expression of MT1-, MT2-, and MT3-MMPs and their correlation with pro-MMP-2 activation, and localization of MMP-2, MT1-MMP, and MT2-MMP in human astrocytic tumors. The sandwich enzyme immunoassay demonstrates that the production levels of pro-MMP-2 in the anaplastic astrocytomas and glioblastomas are significantly higher than that in the low-grade astrocytomas (P<0.05 and P<0.01, respectively), metastatic brain tumors (P<0.05), or normal brains (P<0.01). Gelatin zymography indicates that the pro-MMP-2 activation ratio is significantly higher in the glioblastomas than in other astrocytic tumors (P<0.01), metastatic brain tumors (P<0.01), and normal brains (P<0.01). The quantitative reverse transcription polymerase chain reaction analyses demonstrate that MT1-MMP and MT2-MMP are expressed predominantly in glioblastoma tissues (17/17 and 12/17 cases, respectively), and their expression levels increase significantly as tumor grade increases. MT3-MMP is detectable in both astrocytic tumor and normal brain tissues, but the mean expression level is approximately 50-fold lower compared with that of MT1-MMP and MT2-MMP in the glioblastomas. The activation ratio of pro-MMP-2 correlates directly with the expression levels of MT1-MMP and MT2-MMP but not MT3-MMP. In situ hybridization indicates that neoplastic astrocytes express MT1-MMP and MT2-MMP in the glioblastoma tissues (5/5 cases and 5/5 cases, respectively). Immunohistochemically, MT1-MMP and MT2-MMP are localized to the neoplastic astrocytes in glioblastoma samples (17/17 cases and 12/17 cases, respectively), which are also positive for MMP-2. In situ zymography shows gelatinolytic activity in the glioblastoma tissues but not in the normal brain tissues. These results suggest that both MT1-MMP and MT2-MMP play a key role in the activation of pro-MMP-2 in the human malignant astrocytic tumors and that the gelatinolytic activity is involved in the astrocytic tumor invasion. (+info)
Collagenase-3 (MMP-13) is expressed by tumor cells in invasive vulvar squamous cell carcinomas.
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Collagenase-3 (MMP-13) is a human matrix metalloproteinase specifically expressed by invading tumor cells in squamous cell carcinomas (SCCs) of the head and neck. Here, we have further elucidated the role of MMP-13 in tumor invasion by examining its expression in invasive malignant tumors of the female genital tract. Using in situ hybridization, expression of MMP-13 mRNA was detected in 9 of 12 vulvar SCCs, primarily in tumor cells, but not in intact vulvar epithelium, in cervical SCCs (n = 12), or in endometrial (n = 11) or ovarian adenocarcinomas (n = 8). MMP-13 expression was especially abundant in vulvar carcinomas showing metastasis to lymph nodes and was associated with expression of membrane type 1 MMP by tumor cells and gelatinase-A (MMP-2) by stromal cells, as detected by immunohistochemistry. MMP-13 mRNAs were detected in 9 of 11 cell lines established from vulvar carcinomas and in 4 of 6 cell lines from cervical carcinomas, whereas endometrial (n = 10) and ovarian (n = 9) carcinoma cell lines were negative for MMP-13 mRNA. No correlation was detected between MMP-13 expression and p53 gene mutations in vulvar SCC cell lines. However, MMP-13 expression was detected in 5 of 6 vulvar and cervical SCC cell lines harboring HPV 16 or 68 DNA. These results show that MMP-13 is specifically expressed by malignantly transformed squamous epithelial cells, including vulvar SCC cells, and appears to serve as a marker for their invasive capacity. (+info)
Human diabetic neovascular membranes contain high levels of urokinase and metalloproteinase enzymes.
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PURPOSE: Retinal neovascularization is one of the leading causes of blindness. A crucial event in this process is the remodeling and penetration of the capillary basement membrane by migrating endothelial cells. This process requires proteolysis of basement membrane components by a variety of proteinases. The objective of the present study was to determine the expression of proteinases in human retinal tissues showing active neovascularization. METHODS: Epiretinal neovascular membranes surgically removed from patients with proliferative diabetic retinopathy were analyzed by zymography, and the types and amounts of proteinases present in the tissues were determined. Retinas from nondiabetic donor eyes served as control specimens. RESULTS: Both the high- (54 kDa) and low- (33 kDa) molecular-weight forms of urokinase were present at significantly higher levels in neovascular membranes than in normal retinas. The pro forms of the matrix metalloproteinases (MMP) MMP-2 and MMP-9 were significantly elevated in the neovascular membranes in comparison with levels in normal retinas. In addition, the active forms of these enzymes were present in the membranes, whereas there was no detectable level of the active forms in normal retinas. CONCLUSIONS: Human diabetic neovascular membranes contain high levels of urokinase and MMP. The increased activity of proteinases in the final common pathway of retinal neovascularization indicates that inhibition of these enzymes may be a useful therapeutic target as an alternative approach in the management of proliferative retinopathies. (+info)
Expression of matrix metalloproteinases during murine chorioallantoic placenta maturation.
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A large body of experimental evidence supports the participation of two groups of extracellular proteases, matrix metalloproteinases (MMPs), and plasminogen activators/plasmin, in tissue remodeling in physiological and pathological invasion. In the late mouse placenta, several tissue remodeling and cell invasion processes take place. Spongiotrophoblast migration into maternal decidua, as well as decidual extracellular matrix remodeling require the coordinated action of extracellular proteolytic enzymes. Via Northern and in situ hybridization, we have analyzed the spatio-temporal expression patterns of members of the MMP family (stromelysin-3, gelatinases A and B), as well as their inhibitors TIMP-1, -2 and -3 in late murine placenta (days 10.5 to 18.5 of gestation). Gelatinase activity in placental extracts was assessed by substrate zymography. Gelatinase A and stromelysin-3 were found to be prominently expressed in decidual tissue; shortly after midpregnancy, the decidual expression patterns of gelatinase A and stromelysin-3 became overlapping with each other, as well as with the expression domain of TIMP-2. On the other hand, gelatinase B transcripts were expressed only by trophoblast giant cells at day 10.5, and were downregulated at later stages. TIMP-1 and TIMP-3 transcripts were detected in decidual periphery at day 10.5, while later the expression was restricted to the endometrial stroma and spongiotrophoblasts, respectively. The areas of stromelysin-3 expression were the same (giant trophoblasts) or adjacent (decidua) to those where urokinase (uPA) transcripts were detected, suggesting a possible cooperation between these proteinases in placental remodeling. We generated mice doubly deficient for stromelysin-3 and uPA, and report here that these mice are viable and fertile. Furthermore, these animals do not manifest obvious placental abnormalities, thereby suggesting the existence of compensatory/redundant mechanisms involving other proteolytic enzymes. Our findings document the participation of MMPs and their inhibitors in the process of late murine placenta maturation, and warrant the characterization of other members of the MMP family, like membrane type-MMPs, in this process. (+info)
Role of interleukin 10 and transforming growth factor beta1 in the angiogenesis and metastasis of human prostate primary tumor lines from orthotopic implants in severe combined immunodeficiency mice.
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Transfection of primary human prostate tumor cells (i.e., HPCA-10a, 10b, 10c, and 10d lines) with the transforming growth factor (TGF)-beta1 gene stimulated anchorage-independent growth and promoted tumor growth, angiogenesis, and metastasis after orthotopic implantation in severe combined immunodeficiency mice. In contrast, interleukin (IL)-10 transfected cells or cells cotransfected with these two genes exhibited reduced growth rates and significantly reduced angiogenesis and metastasis after 8, 12, and 16 weeks. Enzyme-linked immunosandwich assays confirmed that the respective tumors expressed elevated levels of TGF-beta1 and IL-10 in vivo. ELISAs further showed that TGF-beta1 expression induced matrix metalloproteinases-2 (MMP-2) expression, whereas IL-10 down-regulated MMP-2 expression while up regulating TIMP-1 in the transfected cells. Also, tumor factor VIII levels correlated with TGF-beta1 and MMP-2 expression and inversely with IL-10 and TIMP-1 levels. More importantly, mouse survival was zero after 4-6 months in mice bearing TGF-beta1- and MMP-2-expressing tumors and increased significantly in mice implanted with IL-10- and TIMP-1-expressing tumors (i.e., to >80% survival). Analysis of the metastatic lesions showed that they expressed TGF-beta1 and MMP-2 but barely detectable levels of IL-10 or TIMP-1, suggesting that IL-10 and TIMP-1 might normally block tumor growth, angiogenesis, and metastasis. (+info)
Residue 2 of TIMP-1 is a major determinant of affinity and specificity for matrix metalloproteinases but effects of substitutions do not correlate with those of the corresponding P1' residue of substrate.
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The unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1.MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys1 and Cys70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP-1 interacts with the S1' specificity pocket of MMP-3, which is a key to substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases. (+info)
Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo.
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Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor. (+info)
Human tissue inhibitor of metalloproteinases 3 interacts with both the N- and C-terminal domains of gelatinases A and B. Regulation by polyanions.
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We compared the association constants of tissue inhibitor of metalloproteinases (TIMP)-3 with various matrix metalloproteinases with those for TIMP-1 and TIMP-2 using a continuous assay. TIMP-3 behaved more like TIMP-2 than TIMP-1, showing rapid association with gelatinases A and B. Experiments with the N-terminal domain of gelatinase A, the isolated C-terminal domain, or an inactive progelatinase A mutant showed that the hemopexin domain of gelatinase A makes an important contribution to the interaction with TIMP-3. The exchange of portions of the gelatinase A hemopexin domain with that of stromelysin revealed that residues 568-631 of gelatinase A were required for rapid association with TIMP-3. The N-terminal domain of gelatinase B alone also showed slower association with TIMP-3, again implying significant C-domain interactions. The isolation of complexes between TIMP-3 and progelatinases A and B on gelatin-agarose demonstrated that TIMP-3 binds to both proenzymes. We analyzed the effect of various polyanions on the inhibitory activity of TIMP-3 in our soluble assay. The association rate was increased by dextran sulfate, heparin, and heparan sulfate, but not by dermatan sulfate or hyaluronic acid. Because TIMP-3 is sequestered in the extracellular matrix, the presence of certain heparan sulfate proteoglycans could enhance its inhibitory capacity. (+info)