Biomedical subjects

M Seiki

Publications and source records attributed to M Seiki.

At least 127 records · Page 7Linked to original sources

PubMed1996

Effects of indomethacin on the production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 by human articular chondrocytes.

OBJECTIVE: To study the action of indomethacin on cartilage catabolic activity by comparing the production of a matrix degrading proteinase and its inhibitor in human articular chondrocyte cultures. METHODS: Matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in conditioned medium from human articular chondrocyte cultures were measured using a one-step sandwich enzyme immunoassay. TIMP-1 mRNA expression was analyzed by Northern blotting using a 0.6 kb cDNA probe for human TIMP-1. RESULTS: Human recombinant interleukin 1 beta (IL-1 beta) increased MMP-3 levels in primary chondrocyte cultures. Indomethacin at 10(-5) M inhibited this IL-1 beta stimulation, but had no effect in the therapeutic range (10(-6)-10(-7) M). Low levels of indomethacin (10(-7) M) significantly increased the production of TIMP-1 by chondrocytes. Synthesis of TIMP-1 appeared to be inhibited by prostaglandin E2 (PGE2), since exogenously added PGE2 reversed the stimulating effect of indomethacin on TIMP-1 production. Northern blot analysis showed that 10(-7) M indomethacin increased TIMP-1 mRNA levels in chondrocytes. CONCLUSION: Our findings indicate that low levels of indomethacin can benefit matrix metabolism by affecting the balance of proteinases to their inhibitors in human articular cartilage.

Blotting, Northern↗

PubMed1995

Intermolecular autolytic cleavage can contribute to the activation of progelatinase A by cell membranes.

Membrane-type matrix metalloproteinase (MT-MMP) messenger RNA and protein expression were shown to be elevated in human fibroblasts following treatment with concanavalin A, coincident with the induction of the ability to process progelatinase A. CHO cells transfected with the cDNA for MT-MMP were able to process both wild type progelatinase A and a catalytically inactive mutant, E375A progelatinase A. Both proenzymes were converted to a 68-kDa intermediate (reducing gels) form, but only the wild type enzyme was processed further to a 66-kDa end product. In contrast, both forms of progelatinase were processed via the 68-kDa intermediate to 66 kDa by concanavalin A-stimulated fibroblasts. Further study of the processing of E375A progelatinase A by plasma membrane preparations from concanavalin A-stimulated fibroblasts showed that addition of active gelatinase A enhanced processing to the mature form. It was concluded that cell membrane-mediated activation of progelatinase A could be via a cascade involving both MT-MMP and intermolecular autolytic cleavage.

Concanavalin A↗

PubMed1995

Activation of the precursor of gelatinase A/72 kDa type IV collagenase/MMP-2 in lung carcinomas correlates with the expression of membrane-type matrix metalloproteinase (MT-MMP) and with lymph node metastasis.

We have identified a novel membrane-type matrix metalloproteinase (MT-MMP) expressed on the cell surface and inducing activation of pro-gelatinase A in vitro. In this study, we further examined the possibility that MT-MMP is the activator of pro-gelatinase A in tumors as well as in vitro. Expression of MT-MMP mRNA was analyzed by Northern blotting in 58 cases of human lung carcinomas. MT-MMP mRNA expression was increased in tumor tissues compared with adjacent normal tissues. The ratio of MT-MMP mRNA levels in tumor/normal tissues (T/N ratio) was 3.19 +/- 1.62 in 29 cases of adenocarcinoma, 3.09 +/- 1.44 in 24 cases of squamous cell carcinoma, 4.40 +/- 0.47 in 3 cases of large cell carcinoma and 3.63 +/- 2.11 in 2 cases of small cell carcinoma, respectively. Activated gelatinase A, as detected by gelatin zymography, was also predominant in tumors compared with normal tissue counterparts, though the difference in mRNA levels was not significant. The activation ratio of gelatinase A in tumor vs. normal tissues correlated well with that of MT-MMP mRNA expression and with lymph node metastases. Our findings suggest that MT-MMP is indeed the tumor-specific activator of pro-gelatinase A in lung carcinomas and is important to initiate invasion of basement membranes.

DNA, Complementary↗

PubMed1995

Thrombin induces the activation of progelatinase A in vascular endothelial cells. Physiologic regulation of angiogenesis.

Angiogenesis requires degradation of vascular basement membrane prior to migration and proliferation of endothelial cells; proteinases are essential ingredients in this process. Because of thrombin's multiple effects on endothelium, we have examined its role in matrix metalloproteinase activation using human umbilical vein endothelial cells. Gelatin zymography of endothelial conditioned media revealed a prominent 72-kDa progelatinase A band. Addition of alpha-thrombin to endothelial cells resulted in the generation of 64 and 62 kDa gelatinolytic bands which is consistent with the activation of progelatinase A; thrombin had no effect in the absence of cells. This effect requires the proteolytic site of thrombin since progelatinase A activation was abolished by specific inhibitors of thrombin. Matrix metaloproteinase inhibitors diminished thrombin-induced activation of progelatinase A. Pretreatment of endothelial cells with excess tissue inhibitor of metalloproteinase-2 or a COOH-terminal fragment of progelatinase A abrogated thrombin-mediated activation of progelatinase A presumably by competing with the COOH terminus of native progelatinase A for interaction with an activator site on endothelial plasma membranes. Although membrane-type matrix metalloproteinase was demonstrated in endothelial cells by Northern and Western blotting, the receptor function of this molecule in thrombin-induced activation of progelatinase A needs to be clarified. Progelatinase A activation did not require intracellular signal transduction events mediated by the thrombin receptor. These data demonstrate that 1) endothelial cells express a novel activation mechanism for progelatinase A, 2) proteolytically active thrombin regulates this activation mechanism, and 3) activation occurs independently of the functional thrombin receptor.

Amino Acid Chloromethyl Ketones↗

PubMed1995

Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library. MT-MMPs form a unique membrane-type subclass in the MMP family.

Membrane-type matrix metalloproteinase (MT-MMP), which we have identified recently, is unique in its transmembrane (TM) domain at the C terminus and mediates activation of pro-gelatinase A on the cell surface (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65; Takino, T., Sato, H., Yamamoto, E., and Seiki, M. (1995) Gene (Amst.) 115, 293-298). In addition to MT-MMP, a novel MMP-related cDNA of 2.1 kilobases was isolated from a human placenta cDNA library. The cDNA contains an open reading frame for a new MMP. The deduced protein composed of 604 amino acids was closely related to MT-MMP in the amino acid sequence (66% homology at the catalytic domains) and has a potential TM domain at the C terminus. Monoclonal antibodies raised against the synthetic peptide recognized a 64-kDa protein as the major product in the transfected cells. TIMP-1 fused with the potential TM domain was localized on the cell surface while native TIMP-1 is in the culture medium. Thus, we called the second membrane-type MMP, MT-MMP-2 and renamed MT-MMP, MT-MMP-1. MT-MMP-1 and -2 are thought to form a distinct membrane-type subclass in the MMP family since all the others are secreted as soluble forms. Like MT-MMP-1, expression of MT-MMP-2 induced processing of pro-gelatinase A (68-kDa in gelatin zymography) into the activated form of 62-kDa fragments through a 64-kDa intermediate form. Expression of MT-MMP-2 mRNA was at the highest levels in the brain where MT-MMP-1 was at the lowest level compared to other tissues. MT-MMP-1 and -2 are thought to be utilized for extracellular matrix turnover on the surface of cells under different genetic controls.

Amino Acid Sequence↗

PubMed1995

Expression of membrane-type matrix metalloproteinase in human gastric carcinomas.

We have examined the implications of membrane-type matrix metalloproteinase (MT-MMP) for activation of the zymogen of MMP-2 (proMMP-2) in human gastric carcinoma. Northern blot analysis demonstrated exclusive expression of MT-MMP in the carcinoma tissues. Immunohistochemically, MT-MMP was colocalized in the carcinoma cells in almost all MMP-2-positive cases (13 of 14 cases). Gelatin zymography of the culture media showed a correlation of the proMMP-2 activation with MT-MMP expression in the carcinoma cells. A microdissection study indicated that proMMP-2 activation is caused only in the carcinoma cell nests that express MT-MMP but not in the normal gastric mucosa. These results are the first demonstration of the MT-MMP-assisted activation of proMMP-2 in the human gastric carcinoma.

Carcinoma↗

PubMed1995

Complex regulation of membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation by concanavalin A in MDA-MB-231 human breast cancer cells.

Matrix Metalloproteinase-2 (MMP-2) is secreted as a zymogen, the activation of which has been associated with metastatic progression in human breast cancer (HBC). Concanavalin A (Con A) has been found to induce activation of MMP-2 in invasive HBC cell lines. Con A effects on the expression of mRNA for membrane-type matrix metalloproteinase (MT-MMP), a newly described cell surface-associated MMP, showed a close temporal correlation with induction of MMP-2 activation. It is surprising that MT-MMP mRNA is constitutively present in the uninduced MDA-MB-231 cell, despite a lack of MMP-2 activation. We have used actinomycin D to demonstrate a partial requirement for de novo gene expression in the induction of MMP-2 activation by Con A in MDA-MB-231 HBC cells. Furthermore, this transcriptional response to Con A appeared to require the continued presence of Con A for its manifestation. The nontranscriptional component of the Con A induction manifests rapidly, is quite substantial, and persists strongly despite actinomycin D abrogation of both constitutive and Con A-induced MT-MMP. Cycloheximide analyses suggest that protein synthesis may be involved in this rapid transcription-independent response. These studies suggest that Con A induces MMP-2-activation in part by up-regulation of MT-MMP expression but has a more complicated mode of action, involving additional nontranscriptional effects, which apparently require protein synthesis.

Breast Neoplasms↗

PubMed1995

Identification of the Tax interaction region of serum response factor that mediates the aberrant induction of immediate early genes through CArG boxes by HTLV-I Tax.

Tax of human T-cell leukemia virus type I (HTLV-I) activates transcription at a CArG box of various immediate early genes such as the proto-oncogene c-fos. To do this, Tax does not directly bind to the CArG box, but instead binds to the CArG binding factor SRF. In this study, we investigated the domain of SRF required for the activation by Tax and studied the role of this domain on transcriptional regulation at the CArG box. Using a fusion protein of SRF with a yeast transcription factor GAL4, the 14 amino acid (aa) portion (aa 422-435) of SRF was identified as the domain required for Tax activation [Tax-responsive region of SRF (TRRS)]. By means of a two hybrid system, we showed that TRRS was essential for the interaction of SRF with Tax in vivo. The over-expression of SRF with a deletion of TRRS inhibited the Tax activation at the CArG box. Thus, TRRS is the domain of SRF that is essential for Tax activation at the CArG box. Unlike to Tax activation, TRRS was not required for TPA (12-o-tetradecanoylphobol-13-acetate) induction at the CArG box, but a TRRS deletion enhanced the basal activity at the CArG box both under serum-starved and TPA-stimulated conditions. These results suggest that TRRS negatively regulates the transcriptional activation function of SRF, and consequently contributes to the low basal activity at the CArG box before TPA induction.

Amino Acid Sequence↗

PubMed1995

Ets-related protein E1A-F can activate three different matrix metalloproteinase gene promoters.

An Ets-related E1A-F has been characterized as an enhancer-binding protein for the adenovirus E1A gene. Here we show, in transient expression assays, that E1A-F can activate three different subclasses of the matrix metalloproteinase gene promoters. Expressions of the chloramphenicol acetyltransferase (CAT) reporter gene under the control of stromelysin, type I collagenase and 92 kD type IV collagenase promoters were increased approximately 10- to 20-fold by co-transfection with the E1A-F expression vector. Activation levels were as much high as those obtained by exogenous expression of AP-1 transcription factor. These results suggest that E1A-F positively regulates transcriptions from matrix metalloproteinase genes that are associated with invasion and metastasis of tumor cells.

Adenovirus E1A Proteins↗

PubMed1995

Cloning of a human gene potentially encoding a novel matrix metalloproteinase having a C-terminal transmembrane domain.

Matrix metalloproteinases (MMPs) play key roles in tissue remodeling during physiological and pathological processes by degrading various extracellular matrix (ECM) components. Although nine distinct MMPs have been characterized by cDNA cloning, there are thought to be more corresponding to the complexity of the ECM. MMP genes expressed in human tissues and cell lines were analyzed by the polymerase chain reaction (PCR) using degenerate primers that corresponded to the conserved amino acid (aa) sequences of the MMPs. One isolated complementary DNA (cDNA) fragment had sequence homology to the reported MMPs, but was otherwise unique. A human placenta cDNA library (Clontech) was screened using the fragment as a probe and a 3.4-kb cDNA fragment containing a long open reading frame (potentially encoding 582 aa) was isolated. The putative gene product had a common domain structure and the conserved sequence of a MMP, but it had a unique transmembrane (TM)-like structure at the C terminus. It should, therefore, be an TM protein, whereas all the other reported MMPs are secretory proteins. Thus, the gene is thought to be the first of a new subclass of MMPs whose products are potentially expressed on the cell surface.

Adenocarcinoma↗

PubMed1995

Induction of M(r) 92,000 type IV collagenase expression in a squamous cell carcinoma cell line by fibroblasts.

A previous investigation (Matsumoto et al., J. Oral Pathol. Med., 18: 498-501, 1989) has shown that the in vitro invasion of a collagen gel by squamous cell carcinoma can be substantially augmented in the presence of fibroblasts. Therefore, we undertook a study to determine if the production of collagenase(s) by a squamous cell carcinoma cell line, UM-SCC-1, was up-regulated by fibroblasts. Cocultivation of UM-SCC-1 cells with MDA-TU-138 fibroblasts, both established from the oral cavity, resulted in a dose-dependent increase in the activity of a M(r) 92,000 gelatinase as shown by zymography. Augmented M(r) 92,000 gelatinase activity was a consequence of the stimulation of the UM-SCC-1 cells by a soluble, fibroblast-derived factor since this effect could be reproduced with fibroblast-conditioned medium but not with glutaraldehyde-fixed fibroblasts. The increased M(r) 92,000 gelatinolytic activity could be accounted for by an increase in M(r) 92,000 type IV collagenase (MMP-9) protein, as demonstrated by Western blotting for this metalloproteinase. Trypsin treatment of the fibroblast-conditioned medium abolished its ability to increase MMP-9 secretion by UM-SCC-1 cells. Furthermore, fractionation of the fibroblast-conditioned medium revealed a M(r) 3,000-10,000 soluble factor(s) which was responsible for the augmented production of MMP-9 by UM-SCC-1 cells. To determine if the increased production of MMP-9, in response to the fibroblasts, was a consequence of increased promoter activity, UM-SCC-1 cells were transiently transfected with a chloramphenicol acetyltransferase reporter driven by the MMP-9 promoter and plated on plastic or on a monolayer of MDA-TU-138 fibroblasts. A 4-5-fold stimulation of MMP-9 promoter activity was observed with UM-SCC-1 cells plated with the MDA-TU-138 fibroblasts, when compared with similarly transfected cells recultured on plastic. In conclusion, we have demonstrated that MMP-9 expression in a squamous cell carcinoma cell line is augmented by a fibroblast-derived protein(s). This finding indicates a role for stromal cells in the regulation of MMP-9 expression in squamous cell carcinoma. The ability of fibroblasts to regulate MMP-9 expression in tumor cells in vitro may explain the observation that the amount of M(r) 92,000 type IV collagenase mRNA in tumor cells is highest at the tumor:stromal interface of resected squamous cell carcinoma.

Carcinoma, Squamous Cell↗

PubMed1995

The C-terminal region of membrane type matrix metalloproteinase is a functional transmembrane domain required for pro-gelatinase A activation.

We identified a new matrix metalloproteinase (membrane type matrix metalloproteinase (MT-MMP)) that has a potential transmembrane (TM) domain at the C terminus and reported its expression on the surface of invasive tumor cells. The expression of MT-MMP induced specific activation of 72-kDa pro-gelatinase A (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65). Thus, MT-MMP on the cell surface is thought to play an important role in various physiological and pathological processes accompanying tissue remodeling. In this study, we demonstrated that the potential TM domain deduced from the amino acid sequence functions as a membrane linker when it is fused to a secretory protein, tissue inhibitor of matrix metalloproteinases-1. The pro-gelatinase A activation function of MT-MMP was abolished by truncation of the TM domain and recovered by fusing the MT-MMP mutant with the TM domain of interleukin 2 receptor alpha-chain. The truncated MT-MMP was released from the cells into the medium and detected as processed or modified forms. In spite of the deletion of the TM domain some portions of the mutant MT-MMP were still retained on the surface of cells. Thus, MT-MMP has an additional device to keep it on the cell surface. The TM domain however, plays an essential role in the pro-gelatinase A activation function of MT-MMP, probably regulating its fine orientation or the localization that is necessary to interact with substrate.

Animals↗

PubMed1995

White matter microglia produce membrane-type matrix metalloprotease, an activator of gelatinase A, in human brain tissues.

Membrane-type matrix metalloprotease (MT-MMP) is an activator of gelatinase A (MMP-2), which has previously been found in carcinoma cells. We examined non-neurological and Alzheimer's disease brain tissues for MT-MMP by immunohistochemistry and in situ hybridization. The anti-MT-MMP antibodies gave positive staining of brain microglial cells in all the brain tissues. Positively stained microglia were found only in the white matter. The cells producing MT-MMP protein were also shown to be white matter microglia. These results provide further evidence that activated gelatinase A, which may be a processing enzyme for degradation of beta-amyloid protein, may be produced in white matter microglia.

Aged↗

PubMed1995

c-Jun, c-Fos and their family members activate the transcription mediated by three 21-bp repetitive sequences in the HTLV-I long terminal repeat.

Transcription of human T-cell leukemia virus type I is regulated by a viral transactivatior Tax, through the 21-bp sequence in the long terminal repeat (LTR). We found that cellular transcription factor AP-1 (c-Jun/c-Fos heterocomplex) bound to the 21-bp sequence. The binding affinity of the complex increased in proportion to the number of the 21-bp sequence, and the transcriptional activation by AP-1 became evident only when the reporters had more than three 21-bp sequences. Thus, AP-1 may play a role in the viral transcription from the LTR with three 21-bp sequences in the absence of Tax, such as in the early stage of the virus infection.

Amino Acid Sequence↗

PubMed1995

[Studies on antiulcer agents. III. Synthesis and antiulcer activity of phenylpropanol derivatives].

It was found that (+/-)ethyl 2-[2-(3-hydroxypropyl)-4-oxocyclohexylidene]-propionate (1), (+/-)ethyl 2-[2-(3-hydroxypropyl)-4,5-dimethoxyphenyl]propionate (2) and (+/-)3-[2-(3-hydroxypropyl)-4,5-dimethoxyphenyl]-2-butanone (3) had excellent antiulcer activities. In order to study structure-activity relationships, (+/-)2-[(3-hydroxypropyl)phenyl]cyclopentanone derivatives (4, 5) and (+/-)2-[(3-hydroxypropyl)phenyl]cyclopentanol derivatives (6, 7) were synthesized and tested for antiulcer activities. As a result, (+/-)2-[2-(3-hydroxypropyl)-4-methoxy-5-(2- piperidinoethoxy)phenyl]cyclopentanone (5k) exhibited potent antiulcer activities.

1-Propanol↗

PubMed1995

[Studies on antiulcer agents. IV. Synthesis and antiulcer activity of phenylpropanol derivatives].

It was found that (+/-)ethyl 2-[2-(3-hydroxypropyl)-4-oxocyclohexylidene-propionate (1), (+/-)ethyl 2-[2-(3-hydroxypropyl)-4,5-dimethoxyphenyl]propionate (2), (+/-)3-[2-(3-hydroxypropyl)-4,5-dimethoxyphenyl]-2-butanone (3) and (+/-)2-[2-(3-hydroxypropyl)-4-methoxy-5-(2- piperidinoethoxy)phenyl]cyclopentanone (4) had potent antiulcer activities. In order to study structure-activity relationships, (+/-)3-[(3-hydroxypropyl)phenoxy]-2-butanone derivatives (5, 6) were synthesized and tested for antiulcer activities. Among them, (+/-)3-[2-(3-hydroxypropyl)-4-methoxy-5-[3-(4- methylpiperidino)propoxy]phenoxy]-2-butanone.3/2 oxalate (6k) was selected as a preferred antiulcer agent.

1-Propanol↗

PubMed1995

[Molecular biology of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), and the regulation of these genes in tumor tissues].

Degradation of the extracellular matrix (ECM) is required for tumor invasion and metastasis. Matrix metalloproteinases (MMPs), which degrade the various ECM components, are thought to play key roles in these processes. The activity of MMPs is strictly controlled at the following three steps, gene expression, proenzyme activation and inhibition by tissue inhibitors of metalloproteinases (TIMPs). Overexpression of MMP genes and ECM degrading activities have reported to be associated with the invasive and metastatic tumor cells. Many researchers have been reported that these phenotypes of tumor cells were inhibited by TIMPs. Thus, the potential of tumor cells to invade into the ECM is regulated by the balance of MMPs and TIMPs.

Collagenases↗

PubMed1995

[Expression of membrane-type matrix metalloproteinase (MT-MMP) and activation of MMP-2 in lung cancer].

In this study, we investigated the correlation between the expression of MMP-2 and lymph node metastasis by analyzing 58 cases of primary lung cancer. Furthermore we studied expression of membrane-type MMP (MT-MMP) which was identified as an activator of MMP-2 and its relation to the activation ratio of MMP-2 in tumor tissues. Activated form of MMP-2 was detected specifically in the tumor tissues by zymography, and the activation ratio was significantly higher in 20 cases of the lymph node metastasis positive group than in other 38 cases. Additionally, northern blott analysis showed that MT-MMP was overexpressed in cancer tissues and that the expression of MT-MMP was closely related to the amount of activated form of MMP-2. These results indicated that MMP-2, which is activated by MT-MMP expressed on the surface of tumor cells, play a role in tumor metastasis by degrading surrounding basement membranes.

Enzyme Activation↗