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Results for “Matrix Metalloproteinases, Membrane-Associated”

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PubMed1996

Matrix metalloproteinase expression during mouse peri-implantation development.

PROBLEM: The purpose of this study was to define the temporal expression and to quantitate the mRNA levels of collagenase, 72 kDa, 92 kDa, and membrane-type matrix metalloproteinase during the peri-implantation period of pregnancy in the mouse uterus. Embryonic expression of 72 kDa and 92 kDa matrix metalloproteinases, as well as interleukin 1 alpha, was also investigated. METHODS: Uterine matrix metalloproteinases were detected using gelatin substrate gel electrophoresis (zymography) and reverse-transcription polymerase chain reaction methodology was used to detect and quantitate different mRNA species in the mouse uterus and blastocyst. RESULTS: Collagenase, 72 kDa, and 92 kDa matrix metalloproteinases are developmentally regulated during the peri-implantation period of pregnancy, but membrane-type matrix metalloproteinase appears to be expressed constitutively. Matrix metalloproteinase mRNA levels have been quantitated and confirm the observed developmental expression patterns. Prominent expression of bot 92 kDa matrix metalloproteinase and interleukin 1 alpha was observed in blastocysts during outgrowth while weak expression of the 72 kDa matrix metalloproteinase was detected. CONCLUSIONS: The date provide evidence of matrix metalloproteinase expression in vivo and substantiate their potential role in tissue remodeling prior to and during blastocyst implantation. Expression of interleukin 1 alpha, 92 kDa, and 72 kDa matrix metalloproteinases suggests that these proteins are important for trophoblast invasion associated with implantation of the early embryo.

Animals↗

PubMed1996

Proteases associated with invadopodia, and their role in degradation of extracellular matrix.

Metastasizing cancer cells invade the extracellular matrix using plasma membrane protrusions (invadopodia) that contact and dissolve the matrix. Evidence suggests that membrane-associated proteases, 170-kD gelatinase (seprase) and Gelatinase A, exert their mechanisms of action on invadopodia. Potential roles that other metallo- and serine-types of membrane proteases, including membrane-type matrix metalloprotease, meprin, dipeptidyl peptidase IV, fibroblast activation protein alpha and guanidinobenzoatase, play in the cell surface proteolysis are also discussed. It is proposed that formation of a structurally and functionally linked protease complex on invadopodia allows the invasion of cancer cells into the extracellular matrix.

Animals↗

PubMed1996

Stromal cell expression of components of matrix-degrading protease systems in human cancer.

Components of matrix-degrading protease systems are in human cancer often expressed by tumour-infiltrating stromal cells. The cellular pattern of expression of these molecules appears to be unique for each type of cancer. In several cases there are similarities with patterns observed in nonmalignant remodelling processes in the same tissue. These findings indicate that the stromal cells actively participate in the process of cancer invasion. The implications of this new paradigm for cancer biology and cancer treatment are discussed.

Collagenases↗

PubMed1996

MT-MMP, the cell surface activator of proMMP-2 (pro-gelatinase A), is expressed with its substrate in mouse tissue during embryogenesis.

Matrix metalloproteinases (MMPs), which degrade the components of the extracellular matrix, are key enzymes involved in the tissue remodeling of multicellular organisms. Since MMPs are secreted as inactive zymogens (pro-MMPs), they have to be activated to function. We identified a membrane-type MMP (MT-MMP) that activated proMMP-2 (pro-gelatinase A = 72 kDa type IV pro-collagenase) and described its expression on the invasive tumor cell surface. In this study we further examined the expression and role of MT-MMP in the activation of proMMP-2 during mouse embryogenesis. Northern blotting demonstrated that MT-MMP expression was increased together with that of MMP-2 and its inhibitor gene, TIMP-2, in embryos depending upon the number of days after gestation, and decreased with maturation after birth. In situ hybridization and immunohistochemistry localized MT-MMP mRNA and protein in the cells of ossifying tissues where both MMP-2 and TIMP-2 were expressed. Activated MMP-2 was detected by gelatin zymography in the lysates prepared from the micro dissected tissues that expressed the three genes. The activation rate of proMMP-2 was proportional to the expression of MMP-2 and MT-MMP. These results indicated that proMMP-2 activation through its activator, MT-MMP, is a physiological system used by organisms to initiate tissue remodeling on the cell surface.

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

[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↗

PubMed1996

Differential expression of membrane-type matrix metalloproteinase and its correlation with gelatinase A activation in human malignant brain tumors in vivo and in vitro.

In this study, we investigated the expression of activated gelatinase A and membrane-type metalloproteinase (MT-MMP) induced by concanavalin A (ConA) in four highly invasive glioma cell lines (UWR2, UWR3, U251MG, and SNB-19). We also examined gelatinase A and MT-MMP expression in human brain tumor tissues in vivo. Gelatin zymography showed that all four cell lines expressed latent progelatinase A (M(r) 66,000). Activated gelatinase A (M(r) 62,000) was induced by ConA in only UWR2 or UWR3 cells. MT-MMP mRNA was present in all four cell lines prior to ConA treatment, and the relative hybridization signals were 1, 0.80, 0.25, and 0.15 in UWR2, UWR3, U251MG, and SNB-19 cells, respectively. These mRNA signals were dramatically increased (2,8-, 5.4-, and 2.2-fold in UWR2, UWR3, and U251MG cells, respectively) following ConA treatment; however, MT-MMP mRNA expression was unchanged in SNB-19 cells. MT-MMP protein was detected in various amounts in the four cell lines, but only after ConA pretreatment. The amount of MT-MMP mRNA was unchanged in SNB-19 after ConA treatment, and the MT-MMP mRNA level in ConA-treated U251MG was lower than in UWR2 and UWR3 without ConA treatment. MT-MMP protein was detected in SNB-19 and U251 cell lines only after ConA treatment. Gelatin zymography of human brain tumor tissues revealed that almost all samples examined contained a latent form of gelatinase A, whereas the activated form of gelatinase A was only seen in metastatic lung adenocarcinomas and malignant astrocytomas, and especially in glioblastomas. MT-MMP mRNA levels were significantly higher in malignant astrocytomas than in low-grade gliomas and normal brain tissues. These results were confirmed by PCR analysis, which showed that MT-MMP mRNA was absent or barely detectable in normal brain white matter but was easily detectable in malignant astrocytomas. Immunohistochemistry of MT-MMP in frozen sections showed that MT-MMP was localized in neoplastic astrocytes of malignant astrocytomas but was undetectable in normal white brain matter. The data indicate that MT-MMP is present in malignant human glial tumors and that MT-MMP expression correlates with expression and activation of gelatinase A during malignant progression in vivo. A direct correlation between the levels of MT-MMP protein and its transcripts was not found in vitro, suggesting that MT-MMP expression in glioma cell lines might be regulated either at the level of transcription message stability or at posttranscription. Altered MT-MMP expression might contribute, in part, to gelatinase A activation, which in turn facilitates invasion of these tumors.

Astrocytoma↗

PubMed1996

Molecular cloning of a novel membrane-type matrix metalloproteinase from a human breast carcinoma.

A new member of the matrix metalloproteinase (MMP) family of enzymes has been cloned from a human breast carcinoma cDNA library. The isolated cDNA contains an open reading frame 1554 bp long, encoding a polypeptide of 518 amino acids. The predicted amino acid sequence displays a similar domain organization as the remaining MMPs, including a prodomain with the activation locus, the zinc-binding site, and the hemopexin domain. In addition, it contains a C-terminal extension, rich in hydrophobic residues and similar in size to those present in the different membrane-type MMPs (MT-MMPs) identified to date. On the basis of these structural characteristics, this novel MMP has been tentatively called MT4-MMP, because it represents the fourth member of this subclass of MMPs characterized mainly by the occurrence of putative transmembrane domain in their amino acid sequences. MT4-MMP also contains a nine-residue insertion between the propeptide and the catalytic domain, which is a common feature of MT-MMPs and stromelysin-3. This amino acid sequence insertion ends with the consensus sequence R-X-R/K-R, which seems to be essential in the activation of these proteinases by furin. Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues revealed that the MT4-MMP gene (MMP-17) is expressed mainly in the brain, leukocytes, the colon, the ovary, and the testis. The expression of MT4-MMP in leukocytes together with its putative membrane localization suggest that this enzyme could be involved in the activation of membrane-bound precursors of growth factors or inflammatory mediators such as tumor necrosis factor-alpha. In addition, MT4-MMP transcripts were detected in all breast carcinomas, as well as in all breast cancer cell lines analyzed in the present work. On the basis of these expression data in breast tumors, a potential role for human MT4-MMP in the tumoral process is also suggested.

Amino Acid Sequence↗

PubMed1996

Activation of human umbilical vein endothelial cell progelatinase A by phorbol myristate acetate: a protein kinase C-dependent mechanism involving a membrane-type matrix metalloproteinase.

Matrix metalloproteinases play an important role in tumor invasion, angiogenesis and inflammatory tissue destruction. The 72-kd gelatinase A is the most widely distributed. Along with the 92-kd gelatinase B, it plays an important role in basement membrane turnover. Gelatinase A is secreted as progelatinase A and, when activated, can cause extracellular matrix destruction. The physiologic mechanism of this activation is not well understood. Based on the importance of endothelial cells in inflammation and cancer, we sought in this study to systematically study the PMA-induced activation of endothelial cell progelatinase A. Using HUVEC, we demonstrated that PMA-induced activation of progelatinase A in these vascular endothelial cells (a) was protein kinase C-dependent as it was blocked by H-7; (b) occurred through cell-mediated events as PMA was unable to activate progelatinase A in a cell-free system and that low dose tissue inhibitor of metalloproteinases-2, but not tissue inhibitor of metalloproteinases-1, totally inhibited PMA-induced activation; (c) was accompanied by an increase in the membrane-type matrix metalloproteinase (MT-MMP). We also found that the combination of PMA and the cytokine tumor necrosis factor-alpha increased HUVEC secretion and activation of gelatinase B. In conclusion, our data show that PMA activation of vascular endothelial cell progelatinase A is a cell membrane event that is at least partially mediated through a PKC-dependent mechanism and is accompanied by an increase synthesis of MT-MMP. These data suggest a role for MT-MMP in the activation of progelatinase A in vascular endothelial cells.

Cell Membrane↗

PubMed1996

[Experimental studies on the cell adhesion molecule E-cadherin and in vitro invasion of endometrial carcinoma cell lines].

The enhancement of the in vitro invasive ability and the morphological changes caused by anti-E-cadherin antibody HECD-1 were investigated by in vitro invasion assay and electron microscopy in three human endometrial carcinoma cell lines. The cell lines were NUE-1 (E-cadherin negative and high in vitro invasive ability), HEC-1BE and HEC-108 (E-cadherin positive and low in vitro invasive ability). In NUE-1 invasive ability was not enhanced by HECD-1, but in HEC-1BE and HEC-108 invasive ability was enhanced to 223 +/- 41.2% and 307 +/- 173% by 5 micrograms/ml HECD-1. Morphologically NUE-1 invaded the extracellular matrix (Matrigel) with a long micro villis. But in HEC-1BE and HEC-108 the villis did not invade the Matrigel, the whole cell invaded it. Together with HECD-1, HEC-1BE and HEC-108 were changed to become similar to the NUE-1 cell line with high invasive ability and the micro villis invaded the Matrigel.

Adenocarcinoma↗

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↗

PubMed1995

A plasma membrane-associated component of ovarian adenocarcinoma cells enhances the catalytic efficiency of matrix metalloproteinase-2.

Several recent investigations have demonstrated that matrix metalloproteinase-2 (MMP-2) binds to the cell surface and undergoes zymogen activation via a plasma membrane-associated activity. The purpose of this study was to determine if association of MMP-2 with the plasma membrane also modulates the catalytic efficiency of the active enzyme. Using density gradient centrifugation, we isolated the plasma membrane fractions of two ovarian adenocarcinoma cell lines, DOV 13 and OVCA' 432, previously described either to express MMP-2 or to express no gelatinolytic metalloproteinases, respectively. While DOV 13 cells contained plasma membrane-associated MMP-2 and OVCA 432 did not, both cell types were able to bind exogenous MMP-2. Furthermore, plasma membrane fractions from these cells significantly enhanced the rate of cleavage of [14C]gelatin I substrate by both MMP-2 tissue inhibitor of metalloproteinases-2 (TIMP-2) complex (2.5-8-fold) and TIMP-2-free MMP-2 (5.9-fold). This stimulatory activity was dose-dependent, soluble in Triton X-100, and abolished by trypsin treatment of the membranes, but was stable to heat treatment. Plasma membrane stimulation of MMP-2 resulted in a 3.8-4.6-fold increase in the catalytic efficiency of gelatinolysis. These data suggest that, in addition to promoting zymogen activation, cell surface binding of MMP-2 may regulate enzyme activity by increasing the rate of substrate cleavage. Via this mechanism, tumor cell types that do not express MMPs (such as OVCA 432) nevertheless may be able to utilize exogenous MMP-2 to mediate proteolysis associated with invasion and metastasis.

Adenocarcinoma↗

PubMed1992

Tumor cell surface-associated binding site for the M(r) 72,000 type IV collagenase.

We have studied the capacity of two human breast adenocarcinoma cells, MDA-MB231 and MCF-7, to bind exogenous M(r) 72,000 type IV collagenase by both morphological and radioreceptor binding assays. By indirect immunofluorescence, staining with a specific anti-M(r) 72,000 type IV collagenase antibody was strongly induced when cells were preincubated with the purified enzyme. Scatchard plot analysis indicated the existence of a binding site for the M(r) 72,000 type IV collagenase with high affinity for both cell lines (Kd = 2 x 10(-9) M). These results are the first demonstration of the existence of a tumor cell membrane-associated putative receptor for a member of the matrix metalloproteinase family, as previously evidenced for the urokinase-type plasminogen activator.

Adenocarcinoma↗

PubMed1992

The matrix metalloproteinases and their inhibitors.

A number of metalloproteinases that degrade the extracellular matrix of connective tissues and two specific tissue inhibitors of metalloproteinases (TIMPs) have now been isolated, characterized, and cloned. Comparison of the enzyme sequences has allowed the delineation of domain structures, and initial studies have been carried out to assess the contribution of these domains to their biochemical and biologic properties, including activation, inhibition by TIMPs, and matrix binding. Such events represent the major levels of extracellular regulation of metalloproteinase activity, which is thought to be an important aspect of their control. Activation is probably a cell surface phenomenon, involving the plasminogen activator cascade or other membrane-associated mechanisms. The inhibitory action of TIMPs is postulated to be as important in activation as in the subsequent regulation of enzyme degradation of the matrix.

Amino Acid Sequence↗

PubMed1996

The soluble catalytic domain of membrane type 1 matrix metalloproteinase cleaves the propeptide of progelatinase A and initiates autoproteolytic activation. Regulation by TIMP-2 and TIMP-3.

It has been proposed that the cell-mediated activation of progelatinase A requires binding of the C-terminal domain of the proenzyme to a membrane-associated complex of the membrane type matrix metalloproteinase MT1-MMP and TIMP-2. Subsequent sequential proteolysis of the propeptide by MT1-MMP and gelatinase A is thought to generate the active form of gelatinase A. We have prepared the proform of the catalytic domain of the MT1-MMP and demonstrated that this may be activated in vitro by trypsin proteolysis to yield a functional proteinase capable of cleaving typical metalloproteinase peptide substrates, gelatin and casein. The active catalytic domain of MT1-MMP was also shown to activate progelatinase A to a fully active form. Using the inactive mutant pro-E375A gelatinase A, we dissected the propeptide processing events that occur. MT1-MMP cleaves the propeptide at the sequence Asn37-Leu38 only. Further cleavage of the mutant enzyme propeptide at Asn80-Tyr81, equivalent to that of the active wild type gelatinase A, could only be effected by addition of gelatinase A to the system. TIMP-1 was essentially unable to prevent MT1-MMP processing of wild type or E375A progelatinase A, whereas TIMP-2 and TIMP-3 were good inhibitors of these events. Analysis of the rate of binding of TIMPs to the catalytic domain of MT1-MMP using kinetic methods showed that TIMP-1 is an extremely poor inhibitor of MT1-MMP. In comparison, TIMP-2 and TIMP-3 are excellent inhibitors, binding more rapidly to the catalytic domain of MT1-MMP than to the catalytic domain of gelatinase A. These data demonstrate the basic mechanism of MT1-MMP action on progelatinase A and the reason for the lack of inhibition by TIMP-1 previously demonstrated in cell-based activation studies.

Amino Acid Sequence↗

PubMed1996

Plasminogen activators, kallikrein-like proteinase and type I and type IV collagenases at various stages of oncogenic transformation.

Comparative studies of membrane-associated, intracellular and secreted activities of serine (uPA, kallikrein-like proteinase) and metalloproteinases (type I and IV collagenases) were carried out on rat embryo fibroblasts, sequentially immortalized and transformed by two different genes. Using this experimental model it was shown that (1) activity of uPA was expressed at the stage of immortalization solely; (2) intracellular and secreted activity of type I and IV collagenases decreased during process of transformation, (3) kallikrein-like proteinase activity was not revealed either in the primary or in transformed cells, (4) Z-Phe-Arg-MCA hydrolysis was the result of the action of cysteine proteinases alone; the increase in this activity was correlated with the stages of oncogenic transformation of fibroblasts.

Animals↗

PubMed1992

Detection and characterization of Theileria sergenti proteinases.

The lysate of Theileria sergenti piroplasms was tested for proteinases using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which substrate was included in gel matrix. Six proteinases of molecular weight 330, 125, 98, 94, 67 and 58 kilodalton (kDa) were detected. From the results of the Triton X-114 phase partition, 330, 125 and 58 kDa proteinases were partitioned into aqueous phase, which indicated that they were not associated with parasite membranes. All these three enzymes were classified into metalloproteinase family because of their sensitivities to metal-ion chelating compounds, ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline. On the other hand, 98 and 94 kDa proteinases were membrane-associated metalloproteinases which were preferentially inhibited by 1,10-phenanthroline. Another metalloproteinase of 67 kDa which was inhibited by EDTA and 1,10-phenanthroline was not associated with parasite membranes. Proteinases of 98 and 94 kDa degraded heat-denatured hemoglobin.

Animals↗