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Biomedical subjects

M Seiki

Publications and source records attributed to M Seiki.

At least 91 records · Page 5Linked to original sources
PubMed1997

Pulmonary metastatic lesion of endolymphatic stromal myosis expresses metastasis-related genes but not invasion-related matrix type metalloproteinase.

A case of endolymphatic stromal myosis (ELSM) with multiple metastasis to lungs was studied. A single biopsy specimen from the lung was analyzed for c-erbB-2, CD44E, and autocrine motility factor receptors (AMFR) mRNA expression, all putatively associated with metastasis. Estrogen and progestin receptors (ER, PR) expression were studied by reverse transcription polymerase chain reactions. Expression of an invasion-related gene, membrane type matrix metalloproteinase (MT-MMP) was also studied. The metastatic lesion showed positive expression of c-erbB-2, CD44E, AMFR, PR and ER expression, whereas no expression of MT-MMP was detected. These results correspond with the present clinical history that is early and multiple lung metastasis but essentially benign in nature and with excellent response to gestagen treatment.

Biopsy↗
PubMed1997

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 (DeltaMT1) 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 alpha1(I) chains and the Gly775-Leu776 and Gly781-Ile782 bonds for alpha2(I) chains. DeltaMT1 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 DeltaMT1 and MMP-1 indicate that DeltaMT1 is 5-7.1-fold less efficient at cleaving type I collagen. On the other hand, gelatinolytic activity of DeltaMT1 is 8-fold higher than that of MMP-1. DeltaMT1 also digests cartilage proteoglycan, fibronectin, vitronectin and laminin-1 as well as alpha1-proteinase inhibitor and alpha2-macroglobulin. The activity of DeltaMT1 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.

Collagen↗
PubMed1997

Increased expression of membrane type 1-matrix metalloproteinase in head and neck carcinoma.

BACKGROUND: Three types of membrane type-matrix metalloproteinases (MT-MMPs) have been identified as activators of pro-MMP2 (gelatinase A/72-kilodalton Type IV collagenase), which is believed to be crucial for tumor invasion and metastasis. MT1-MMP has been shown to be widely expressed in various tissues and to be overexpressed in lung and gastric carcinomas. METHODS: Activation of pro-MMP2 was examined in head and neck squamous cell carcinoma (HNSCC) by gelatin zymography. The expression of the MT1-MMP mRNA transcript was studied by Northern hybridization and that of MT1-MMP protein by immunohistochemical staining with a monoclonal antibody against MT1-MMP. RESULTS: Activation of pro-MMP2 was observed in ten of ten HNSCC tissues. Increased expression of the MT1-MMP mRNA transcript was also detected in eight of eight of these tissues. MT1-MMP positive cells were detected at the tumor cell lesions in the majority of these carcinoma tissues by immunohistochemical staining (24 of 27). MT1-MMP expression was more intense in moderately and well differentiated tumors than in poorly differentiated ones (P < 0.05). CONCLUSIONS: These results suggest that MT1-MMP is involved in the activation of pro-MMP2 in HNSCC. These findings suggest that MT1-MMP is significant not only as a tumor marker but as a new target for chemotherapy against HNSCC.

Carcinoma, Squamous Cell↗
PubMed1997

Dual expression of matrix metalloproteinase-2 and membrane-type 1-matrix metalloproteinase in fibrotic human livers.

We have previously reported increased expression of matrix metalloproteinase-2 (MMP-2) using a rat model of liver fibrosis. However we did not clarify how the precursor of MMP-2 (proMMP-2) was activated. Therefore, we used human liver specimens with chronic hepatitis (CH) and liver cirrhosis (LC) to examine expression of membrane-type-1-MMP (MT1-MMP), which has recently been determined to activate proMMP-2. Northern hybridization studies showed a 5.4- and 1.4-fold increase in MMP-2 expression in CH and LC, respectively, as compared with normal liver. MT1-MMP gene expression simultaneously increased 4.0- and 1.4-fold in CH and LC, respectively. In situ hybridization using 35S-cRNA probes of MMP-2 and MT1-MMP showed prominent silver granules in elongated cells found in the lobules, periportal areas, and fibrous septa of CH and LC samples. These elongated cells expressed alpha-smooth muscle actin by immunohistochemistry. Immunoelectron microscopic examination localized MMP-2 and MT1-MMP to the rough endoplasmic reticulum of stellate cells located in the lobules and periportal areas, or to fibroblasts in the fibrous septa, suggesting that MMP-2 and MT1-MMP were produced by these cells. In addition, cytoplasmic and membranous immunodeposits of both MMPs were found in endothelial cells, Kupffer cells, capillary endothelial cells, and lymphocytes, indicating that activation of proMMP-2 occurs locally. Increased expression of MMP-2 and MT1-MMP was detected in CH and LC, while dual over-expression was found in stellate cells and fibroblasts, possibly resulting in the increase of active MMP-2 in and around these cells. These findings suggest that activated MMP-2 may remodel liver parenchyma during the process of liver fibrosis.

Adult↗
PubMed1997

Flow injection analysis for measurement of activity of matrix metalloproteinase-7 (MMP-7).

A simple and convenient method for measuring the activity of a recombinant human matrix metalloproteinase 7 (MMP-7, matrilysin) was developed by flow injection analysis (FIA). For this method, purified recombinant MMP-7 zymogen expressed in E. coli and the substrate peptide (MOCAc-Pro-Leu-Gly-Leu-A2pr(DNP)-Ala-Arg-NH2) were used. Following the incubation of substrate peptide with activated r-proMMP-7, the resulting fluorescent product peptide (MOCAc-Pro-Leu-GLY) was monitored with a fluorescence detector (lambda ex 328 mm, lambda em 393 mm) without chromatographic separation. In this FIA system, the analysis time is 2 min and the standard curve is linear from 5 to 100 pmol of the product peptide injected. In order to use this FIA system as a method for screening inhibitors against MMP-7, the effects of CaCl2, EDTA and of the tissue inhibitor of metalloproteinase-1, and -2, were tested. A synthetic PRCGXPD-containing peptide (BS-10) was also observed to inhibit MMP-7 activity, with an IC50 value of 104 microM. Thus, it was concluded that the activity of r-MMP-7 can be reliably measured by the proposed system. Furthermore, to confirm the utility of this FIA system as a screening method, the inhibitory activity of the MMP-related substance in Joro spider (Nephilia clavata) venom was measured by this method. This inhibitory activity was observed in an extract of a venom diluted 1000-fold. Thus, the FIA method is not only simple and quick, but also sensitive enough to screen and analyze the inhibitory properties of a large number of test compounds.

Enzyme Inhibitors↗
PubMed1997

Induction of membrane-type matrix metalloproteinase 1 (MT1-MMP) expression in human fibroblasts by breast adenocarcinoma cells.

Membrane-type matrix metalloproteinase 1 (MT1-MMP) has been recently described as an activator of proMMP-2 (MMP-2) which is involved in tumor invasion. We have shown by in situ hybridization that MT1-MMP is produced by stromal cells in close contact to preinvasive and invasive tumor cells of breast carcinomas. Of particular interest was the observation that some fibroblasts express this enzyme in focal areas in preinvasive lesions, suggesting that particular tumor cells may stimulate fibroblasts to produce MT1-MMP. We have therefore compared the ability of two different breast cancer cell lines, one non-invasive (MCF7) and one invasive (MDA-MB-231) to stimulate MT1-MMP production in human fibroblasts with consequent proMMP-2-activation. The MDA-MB-231 conditioned medium induced MT1-MMP mRNAs in human fibroblasts and a parallel activation of proMMP-2 whereas MCF7 conditioned medium did not have any effect. These results suggest the existence of soluble factor(s) secreted by invasive or some preinvasive breast tumor cells which stimulate fibroblasts to produce and activate MMPs, and emphasize the cooperation between cancer and stromal cells in tumor invasion.

Blotting, Northern↗
PubMed1997

MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells.

We have previously reported that human breast carcinoma (HBC) cell lines expressing the mesenchymal intermediate filament protein vimentin (VIM+) are highly invasive in vitro, and highly metastatic in nude mice when compared to their VIM- counterparts. Since only VIM+ cell lines can be induced to activate matrix metalloproteinase-2 (MMP-2) upon stimulation with Concanavalin A (Con A), we have examined here membrane type 1 MMP (MT1-MMP), a cell surface activator of MMP-2. Northern analysis reveals baseline expression of MT1-MMP in five of the six VIM+ cell lines studied (MDA-MB-231, MDA-MB-435, BT-549, Hs578T, MCF-7(ADR)), each of which showed variable activation of exogenous MMP-2 after treatment with Con A. In contrast, the four VIM-, poorly invasive HBC cell lines studied (MCF-7, T47D, MDA-MB 468, ZR-75-1) lacked baseline MT1-MMP mRNA expression, and showed no induction of either MT1-MMP expression or MMP-2-activation with Con A. Such differential MT1-MMP expression was confirmed in vivo using in situ hybridization analysis of nude mouse tumor xenografts of representative cell lines. Western analysis of the MDA-MB-231 cells revealed baseline membrane expression of a 60 kDa species, which was strongly induced by Con A treatment along with a weaker band co-migrating with that from MT1-MMP-transfected COS-1 cells (63 kDa), presumably representing latent MT1-MMP. MT1-MMP immunofluorescence strongly decorated Con A-stimulated MDA-MB-231 cells in a manner consistent with membranous staining, but did not decorate the unstimulated MDA-MB-231 cells or MCF-7 cells under either condition. Collectively, the results suggest the constitutive production of active MT1-MMP which is unavailable for either MMP-2 activation or immuno-decoration until Con A treatment. Since VIM expression arises by virtue of the so-called epithelial to mesenchymal transition (EMT) in invasive embryonic epithelia, we propose that this represents a major metastasis mechanism in breast carcinomas. MT1-MMP on the surface of such 'fibroblastoid' carcinoma cells may mediate a paracrine loop for the utilization of stromally produced MMP-2, and contribute to the poorer survival associated with VIM+ breast carcinomas.

Animals↗
PubMed1997

Cell-cell contact down-regulates expression of membrane type metalloproteinase-1 (MT1-MMP) in a mouse mammary gland epithelial cell line.

Membrane type matrix metalloproteinase (MT-MMP), which possesses a C-terminal transmembrane domain, is expressed on the cell membrane (Sato et al., 1994, Nature 370: 61-65). It was suspected, therefore, that the expression of MT-MMPs might be regulated by cell-cell interactions. We examined the patterns of MT1-MMP expression in a mouse mammary gland epithelial cell line, HC11, which is capable of responding to prolactin in vitro. HC11 cells form well-differentiated monolayer of cuboidal epithelium at confluence. During the log growth phase, cells which are well dispersed and seemingly migrating actively, or located at the periphery of small colonies, reacted strongly with an anti-MT1-MMP antibody, whereas no MT1-MMP immunoreactivity was detected in the cells which established cell-cell contact with adjacent cells. At confluence, the HC11 cells lost MT1-MMP immunoreactivity completely. Northern blot analysis revealed that MT1-MMP mRNA is present at a high level in HC11 cells during the log phase of growth. Although MT1-MMP immunoreactivity disappeared by the 1st day confluence was reached, the decline of MT1-MMP mRNA levels started only a few days later. The discrepancy in the timing of decrease of MT1-MMP protein and that of the transcripts suggests the presence of translational control mechanisms for MT1-MMP expression during cell-cell interaction.

Animals↗
PubMed1997

Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma.

We have previously demonstrated that fibroblasts and invasive human breast carcinoma (HBC) cells specifically activate matrix metalloproteinase-2 (MMP-2) when cultured on 3-dimensional gels of type I collagen but not a range of other substrates. We show here the constitutive expression of membrane-type 1 (MT1)-MMP in both fibroblasts, and invasive HBC cell lines, that have fibroblastic attributes presumably acquired through an epithelial-to-mesenchymal transition (EMT). Treatment with collagen type I increased the steady-state MT1-MMP mRNA levels in these cells but did not induce either MT1-MMP expression or MMP-2 activation in noninvasive breast carcinoma cell lines, which retain epithelial features. Basal MT3-MMP mRNA expression had a pattern similar to that of MT1-MMP but was not up-regulated by collagen. MT4-MMP mRNA was seen in both invasive and noninvasive HBC cell lines and was also not collagen-regulated, and MT2-MMP mRNA was not detected in any of the HBC cell lines tested. These data support a role for MT1-MMP in the collagen-induced MMP-2-activation seen in these cells. In situ hybridization analysis of archival breast cancer specimens revealed a close parallel in expression of both collagen type I and MT1-MMP mRNA in peritumoral fibroblasts, which was correlated with aggressiveness of the lesion. Relatively high levels of expression of both mRNA species were seen in fibroblasts close to invasive tumor nests and, although only focally, in certain areas close to preinvasive tumors. These foci may represent hot spots for local degradation and invasive progression. Collectively, these results implicate MT1-MMP in collagen-stimulated MMP-2 activation and suggest that this mechanism may be employed in vivo by both tumor-associated fibroblasts and EMT-derived carcinoma cells to facilitate increased invasion and/or metastasis.

Breast Neoplasms↗
PubMed1997

Membrane-type matrix metalloproteinases (MT-MMPs) in cell invasion.

Activated gelatinase A is reportedly associated with tumor spread. We identified a novel matrix metalloproteinase that localizes on the cell surface and mediate the activation of progelatinase A. Thus, this progelatinase A activator was named membrane-type matrix metalloproteinase (MT1-MMP). Following the first-discovery of MT1-MMP, two other MT-MMPs which can activate progelatinase A were identified (MT2- and MT3-MMP, respectively). Among these three MT-MMPs, MT1-MMP is most often overexpressed in malignant tumor tissues, including lung and stomach carcinomas that contain activated gelatinase A. This suggests that MT1-MMP is most closely associated with the activation of progelatinase A in these tumor tissues. The expression of MT1-MMP also induced binding of gelatinase A to the cell surface by functioning as a receptor. The cell surface localization of proteinases has advantages over pericellular proteolysis. MT1-MMP and its family may play a central role in the cell surface localization and activation of progelatinase A and via this mechanism, tumor cells use exogenous progelatinase A to mediate the proteolysis associated with invasion and metastasis.

Chromosome Mapping↗
PubMed1997

v-Rel activates the proto-oncogene c-Jun promoter: a correlation with its transforming activity.

v-Rel is a transforming protein of the reticuloendotheliosis virus, and is a transcription factor regulating various cellular genes. We found that v-Rel activates the promoter of the proto-oncogene c-jun in a transient transfection assay system. Moreover, the expression of endogenous c-jun was augmented in cells expressing exogenous v-Rel, but not c-Rel. The transcriptional activities of v-Rel to the tested promoters containing the kB-site are lower than that of c-Rel, but that to the c-jun promoter was much higher than that of c-Rel. The N-terminal DNA binding domain of v-Rel, which is responsible for its high transforming activity of v-Rel was also responsible for the high transcriptional activity to the c-jun promoter. Thus, the activity of v-Rel upon the c-jun promoter correlates well with its transforming ability. Since c-Jun plays pivotal roles on cell proliferation in various types of cells, the activation of c-jun expression by v-Rel may be an essential step for the oncogenic transformation caused by v-Rel.

Binding Sites↗
PubMed1997

Expression of membrane-type 1 matrix metalloproteinase and activation of progelatinase A in human osteoarthritic cartilage.

Matrix metalloproteinases (MMPs) are expressed in osteoarthritic (OA) cartilage and are thought to be involved in the degradation of cartilage extracellular matrix (ECM). Among these proteinases, MMP-2 (gelatinase A) demonstrates a wide range of substrate specificity against the ECM present in cartilage. Although MMP-2 expression increases in OA cartilage, the activation mechanism of the corresponding zymogen (pro-MMP-2) in cartilage is unknown. In this study, we examined the expression pattern of membrane-type 1 MMP (MT1-MMP) in human OA articular cartilage and its correlation with the activation of pro-MMP-2. Immunohistochemical studies demonstrate that MT1-MMP localizes to the chondrocytes in the superficial and transitional zones in all of the samples examined directly correlating with cartilage degradation. Reverse transcription polymerase chain reaction confirmed the predominant expression of MT1-MMP mRNA in the OA cartilage. In situ hybridization revealed the site of expression of MT1-MMP in OA cartilage to be the chondrocytes. Through gelatin zymography and a sandwich enzyme immunoassay it was demonstrated that OA cartilage explants secrete significantly higher levels of pro-MMP-2 than normal samples. Pro-MMP-2 activation was enhanced in the OA cartilage samples and correlated with MT1-MMP expression in the cartilage. Plasma membranes prepared from cultured chondrocytes with MT1-MMP expression and those directly isolated from OA cartilage could activate pro-MMP-2. MT1-MMP gene expression in cultured chondrocytes was induced by treatment with interleukin-1 alpha and/or tumor necrosis factor-alpha. These data suggest that cytokine-induced MT1-MMP in the chondrocytes may play a key role in the activation of pro-MMP-2 in the OA articular cartilage, leading to cartilage destruction through ECM degradation.

Aged↗
PubMed1997

Suppression of matrix metalloproteinase-3 synthesis by interleukin-4 in human articular chondrocytes.

OBJECTIVE: To evaluate the effect of interleukin-4 (IL-4) on IL-1 induced matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinase-1 (TIMP-1) production by human articular chondrocytes. METHODS: Monolayer cell culture of chondrocytes was obtained from human articular cartilage from patella within 24 h after death. MMP-3 and TIMP-1 protein levels were determined by ELISA. MMP-3 activity was assayed as caseinase activity. Amounts of MMP-3 and TIMP-1 mRNA were measured by Northern blot analysis. RESULTS: IL-4 suppressed IL-1 stimulated MMP-3 protein and enzyme activity. Moreover, IL-4 suppressed IL-1 induced MMP-3 mRNA. In contrast, IL-4 did not alter the level of TIMP-1 protein and mRNA. CONCLUSION: IL-4 may be implicated as a protective mediator of joint destruction seen in inflammatory arthritis.

Adult↗
PubMed1996

Expression of membrane-type matrix metalloproteinase 1 (MT1-MMP) in tumor cells enhances pulmonary metastasis in an experimental metastasis assay.

Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a member of the recently identified unique membrane-type subgroup in the matrix metalloproteinase (MMP) family. MT1-MMP has proteolytic activity against components in the extracellular matrix and activates progelatinase A (72-kDa type IV procollagenase/proMMP-2) on the cell surface. Because MT1-MMP is frequently expressed in a variety of tumors, we examined its contribution to their metastatic potential. The mouse lung carcinoma cell line Madison 109 was transiently transfected with a MT1-MMP expression plasmid and inoculated into the tail vein of BALB/c mouse. Fate of the transfected cells was monitored by the neo(r) gene in the plasmid using the quantitative PCR method. The survival rate of the parental cells in lung was 0.7% of the inoculated cells. It was increased by 3-fold with the MT1-MMP transfected cells and the number of the lung nodules increased accordingly. Immunostaining of the consecutive tissue sections revealed that lung nodules expressing MT1-MMP were positive for gelatinase A as well, whereas MT1-MMP-negative cells were not stained for gelatinase A at all. Thus, MT1-MMP-expressing cells acquire specific ability to bind exogenous progelatinase A.

Animals↗
PubMed1996

Dual over-expression pattern of membrane-type metalloproteinase-1 in cancer and stromal cells in human gastrointestinal carcinoma revealed by in situ hybridization and immunoelectron microscopy.

Membrane-type metalloproteinase-I (MTI-MMP) is a transmembrane metalloproteinase, which activates pro-gelatinase A. There has been disagreement as to whether the cell types expressing MTI-MMP are cancer cells or stromal fibroblasts. Using human gastrointestinal carcinomas, the present study disclosed the tissue localization of MTI-MMP mRNA by in situ hybridization and ultrastructural localization of its protein by immunoelectron microscopy. In normal colon and stomach tissues, MTI-MMP mRNA and protein were negative or faintly positive both in epithelial cells and in stromal fibroblasts, except in the fundic gland of the stomach, which showed the positivity for MTI-MMP. In contrast, gastrointestinal cancer tissue showed over-expression of MTI-MMP mRNA and protein both in cancer cells and in stromal cells (fibroblasts). Stromal fibroblasts also expressed mRNA for gelatinase A and type-I procollagen. Double immunohistochemistry revealed that macrophages were also positive for MTI-MMP. Immunoelectron microscopy showed that MTI-MMP was localized along the plasma membrane of cancer cells and macrophages and in rough endoplasmic reticulum of fibroblasts. The present study reveals a dual over-expression pattern of MTI-MMP both in cancer cells and in stromal fibroblasts; the expression in cancer cells may be related to the invasive growth, whereas that in fibroblasts may be related to the tissue remodeling process caused by invasive growth of cancer cells.

Carcinoma↗
PubMed1996

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.

Arginine↗