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PubMed2000

Distribution pattern of matrix metalloproteinases 1, 2, 3, and 9, tissue inhibitors of matrix metalloproteinases 1 and 2, and alpha 2-macroglobulin in cases of generalized AA- and AL amyloidosis.

Matrix metalloproteinases (MMPs) degrade basement membranes and connective tissue and play an essential role in the homeostasis of the extracellular matrix which is disrupted by the deposition of amyloid. This immunohistochemical study investigated the distribution pattern of matrix metalloproteinases (MMP-1, -2, -3, and -9) and their inhibitors [alpha 2-macroglobulin (alpha 2-M), tissue inhibitors of MMPs (TIMP)-1, and TIMP-2] in human AA- and AL amyloid deposits. Specimens of liver, kidney, and spleen from 22 autopsy cases were investigated. Nine patients had suffered from generalized AA amyloidosis, eight from generalized AL amyloidosis, and five from rheumatoid arthritis or tuberculosis with no histological evidence of amyloid. In all amyloidotic and non-amyloidotic patients, each protease and protease inhibitor was detected in almost every organ investigated. In the amyloidotic cases, there was no indication that a specific protease or protease inhibitor was absent or expressed, but a difference was observed in their spatial distribution patterns. The most noticeable difference was found in immunostaining of amyloid. Only MMP-1, -2, and -3, and alpha 2-M were present in AA amyloid deposits, and only TIMP-1 and TIMP-2 were found in deposits of AL amyloid. This is the first study to show that MMP-1, -2, and -3 are present in AA amyloid deposits. They may be involved in tissue remodeling or in proteolysis of the precursor and fibril proteins.

Adult↗

PubMed2000

Expression and purification of soluble and inactive mutant forms of membrane type 1 matrix metalloproteinase.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteinase and a cell-surface receptor and activator of gelatinase A in normal and neoplastic cells. We have expressed and purified a soluble deletion mutant of MT1-MMP lacking the transmembrane and cytoplasmic domains and an inactive mutant of the soluble MT1-MMP, where the active-site glutamic acid(240) was substituted by alanine (E240A). A baculovirus transfer vector coding for amino acids 21-539 of MT1-MMP (DeltaTM) and a similar vector coding for the mutation (E240ADeltaTM) were constructed for expression in insect cells. Both DeltaTM and E240ADeltaTM were secreted to the culture medium of infected High Five insect cells. They were then purified by cation-exchange followed by gel-filtration chromatography. DeltaTM was able to cleave denatured type I collagen and fibronectin and activate MMP-2/gelatinase-A, while E240ADeltaTM had only low proteolytic activity against denatured collagen I. The current expression and purification protocol should prove useful for the production of large amounts of enzymatically active soluble MT1-MMP.

Amino Acid Substitution↗

PubMed2003

Glycosylation broadens the substrate profile of membrane type 1 matrix metalloproteinase.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a collagenolytic enzyme that has been implicated in normal development and in pathological processes such as cancer metastasis. The activity of MT1-MMP is regulated extensively at the post-translational level, and the current data support the hypothesis that MT1-MMP activity is modulated by glycosylation. Enzymatic deglycosylation, site-directed mutagenesis, and lectin precipitation assays were used to demonstrate that MT1-MMP contains O-linked complex carbohydrates on the Thr(291), Thr(299), Thr(300), and/or Ser(301) residues in the proline-rich linker region. MT1-MMP glycoforms were detected in human cancer cell lines, suggesting that MT1-MMP activity may be regulated by differential glycosylation in vivo. Although the autolytic processing and interstitial collagenase activity of MT1-MMP were not impaired in glycosylation-deficient mutants, cell surface MT1-MMP-catalyzed activation of pro-matrix metalloproteinase-2 (proMMP-2) required proper glycosylation of MT1-MMP. The inability of carbohydrate-free MT1-MMP to activate proMMP-2 was not a result of defective MT1-MMP zymogen activation, aberrant protein stability, or inability of the mature enzyme to oligomerize. Rather, our data support a mechanism whereby glycosylation affects the recruitment of tissue inhibitor of metalloproteinases-2 (TIMP-2) to the cell surface, resulting in defective formation of the MT1-MMP/TIMP-2/proMMP-2 trimeric activation complex. These data provide evidence for an additional mechanism for post-translational control of MT1-MMP activity and suggest that glycosylation of MT1-MMP may regulate its substrate targeting.

Amino Acid Sequence↗

PubMed1999

Matrix-metalloproteinases 1, 2, and 3 and their tissue inhibitors 1 and 2 in benign and malignant breast lesions: an in situ hybridization study.

Invasive growth requires degradation of extracellular matrix. Altered expression of matrix degrading enzymes may indicate an increased potential for invasive growth. We determined the expression patterns of matrix-metalloproteinases (MMP)-1, -2, and -3 and of the tissue inhibitors of metalloproteinases (TIMP)-1 and -2 by in situ hybridization with isotopically labeled RNA probes in normal breast tissue (n=6), fibrocystic disease (n=20), five cases of which contained radial scars, lobular carcinoma in situ (CLIS; n=5), ductal carcinoma in situ (DCIS; n=9) and invasive carcinomas (n=24). Only a few cells displayed MMP-1- and MMP-2-specific labeling in normal breast tissue and fibrocystic disease. Noninvasive ductal carcinomas showed elevated MMP-2 transcript levels in peritumor stromal cells in the absence of significant MMP-1 specific signals. In general, compared with adjacent normal breast tissue, a gradual increase of MMP-2 was found in noninvasive to invasive cancers. Invasive ductal and lobular carcinomas displayed co-expression of MMP-1 and MMP-2 by stromal cells, mainly of the invasion front, with high signal intensity particularly in high-grade invasive carcinomas. Tumor cells and peritumor stroma showed low MMP-3 transcript levels, especially in medullary carcinomas. TIMP-1 and -2 transcript levels were increased in invasive carcinomas correlating with the histological grade. These RNA expression patterns suggest an increased invasive potential in breast carcinomas even prior to histologically overt invasive growth.

Adenocarcinoma↗

PubMed1997

Matrix metalloproteinases 1 and 3, tissue inhibitor of metalloproteinase-1 and the complex of metalloproteinase-1/tissue inhibitor in plasma of patients with prostate cancer.

We analyzed blood plasma concentrations of matrix metalloproteinase-1 and -3 (MMP-1; MMP-3), the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the complex MMP-1/TIMP-1, and looked for any correlation with prostate cancer stage. These components were measured by ELISA tests specific for these proteins in healthy male controls (n = 35), and in patients with benign prostatic hyperplasia (BPH; n = 29), with prostate cancer (PCa) without metastasis (T2,3pN0M0; n = 29) and with PCa with metastatic disease (T2,3,4pN1,2M1; n = 18). Mean values of MMP-1 and of the complex MMP-1/TIMP-1 were not different among the 4 groups studied. The mean MMP-3 and especially TIMP-1 concentrations were significantly higher in PCa patients with metastases compared with controls, BPH and PCa patients without metastases. Ten of these 18 patients had TIMP-1 concentrations higher than the upper reference limit. TIMP-1 concentrations were correlated with staging but not with grading. Our results point towards plasma TIMP-1 concentration as a potential marker of malignant progression of PCa.

Adult↗

PubMed2000

Front-cell-specific expression of membrane-type 1 matrix metalloproteinase and gelatinase A during cohort migration of colon carcinoma cells induced by hepatocyte growth factor/scatter factor.

Migration of tumor cells is usually assessed as single cell locomotion in vitro using Boyden chamber type assays. In vivo, however, carcinoma cells frequently invade the surrounding tissue as coherent clusters or nests of cells. We have called this type of movement "cohort migration" and developed a two-dimensional in vitro cohort migration model, in which human rectal well-differentiated adenocarcinoma cells (L-10) migrate from piled-up cell islands as coherent sheets of cells when stimulated with hepatocyte growth factor/scatter factor. In this study, we examined whether there is a cohort migration-specific way of expression of matrix metalloproteinases (MMP) and whether degradation of extracellular matrix is necessary for this type of migration. Production of membrane-type 1-MMP (MT1-MMP) and gelatinase A (MMP-2) by L-10 cells was demonstrated by gelatin zymography, immunoblotting, and reverse transcription-PCR. When cohort migration was induced with hepatocyte growth factor/scatter factor, MT1-MMP and MMP-2 were immunolocalized predominantly in the leading edges of the front cells of migrating cell sheets, with the following cells being negative. In addition, during the cohort migration on gelatin-coated substratum, the gelatin matrix was degraded by the cells, in a very organized manner, causing radially arrayed lysis of gelatin matrix at the sites of leading edges. BB94, a synthetic inhibitor specific to MMPs, tissue inhibitor of metalloproteinases-1 and -2, and the COOH-terminal hemopexin-like domain of MMP-2 inhibited the migration on gelatin matrix. Thus, these data demonstrate that gelatin matrix is reorganized to suit cell migration via leading-edge-of-front-cell-specific localization of MT1-MMP and MMP-2 during cohort migration and suggest that the reorganization is essential for this type of migration.

Adenocarcinoma↗

PubMed2000

Immunohistochemical expression of matrix metalloproteinases 1 and 2 (MMP-1 and MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in ruptured and non-ruptured tubal ectopic pregnancies.

OBJECTIVE: Recent evidence suggests that matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are crucial for trophoblast implantation in normal pregnancy. To evaluate the expression of MMP-1, MMP2, and the tissue inhibitor of MMP-2 (TIMP-2) along the invasive pathway of trophoblast in ruptured and non-ruptured tubal ectopic pregnancies, we performed a retrospective immunohistochemical study. METHODS: In 15 tissue specimens of patients with ruptured (N = 7) and non-ruptured (N = 8) first trimester tubal ectopic pregnancies who underwent laparoscopic salpingectomy, immunohistochemical staining against MMP-1, MMP-2, and TIMP-2 was performed. Serial paraffin sections were photographed and digitized for a computerized quantitative image analysis. Mean percentages of positive stained areas by MMP-1, MMP-2, and TIMP-2 antibodies in the extravillous trophoblast were determined for ruptured and non-ruptured tubal ectopic pregnancies and compared. RESULTS: In our 15 tissue specimens of ectopic pregnancies MMP-1 and TIMP-2 were found to be more prominent in the immunohistochemical distribution pattern than MMP-2. However, no statistically significant difference could be detected between the mean percentages of positive stained area by MMP-1, MMP-2, and TIMP-2 antibodies in ruptured and non-ruptured tubal pregnancies. DISCUSSION: For the first time, we measured the comparative immunohistochemical expression of MMP-1, MMP-2, and TIMP-2 in ruptured and non-ruptured tubal ectopic pregnancies. Although our results did not show any statistically significant difference between ruptured and non-ruptured tubal ectopic pregnancies, we conclude that MMP-1, MMP-2, and TIMP-2 are functionally involved in the highly proliferative early first part of ectopic implantation.

Adult↗

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↗

PubMed2003

Restricted expression of membrane type 1-matrix metalloproteinase by myofibroblasts adjacent to human breast cancer cells.

The membrane type-1 matrix metalloproteinase (MT1-MMP), a protease originally identified in breast carcinoma, is characterized by its capacity to activate other MMPs (MMP-2 and MMP-13) and to degrade extracellular matrix. Our study was undertaken to localize and identify the MT1-MMP expressing cells in human breast adenocarcinomas. A textural analysis of images obtained by immunohistochemistry and in situ hybridization showed precisely the co-expression of alpha smooth muscle actin (alphaSM actin) and MT1-MMP in myofibroblasts. MT1-MMP expression is confined to myofibroblasts in close contact with tumor cells. In sharp contrast, the expression of MMP-2 was more widely distributed in both alphaSM actin positive and negative cells close to and at distance from cancer cell clusters. Our in vitro observations are consistent with the higher level of MT1-MMP expression and of MMP-2 activation observed in alphaSM actin positive fibroblasts derived from breast tumors, as compared to normal breast fibroblasts. Collectively, these results implicate myofibroblasts as major producer of MT1-MMP in breast cancer and emphasize the importance of stromal-epithelial cell interactions in their progression.

Actins↗

PubMed2003

Novel processing of beta-amyloid precursor protein catalyzed by membrane type 1 matrix metalloproteinase releases a fragment lacking the inhibitor domain against gelatinase A.

In various mammalian cell lines, beta-amyloid precursor protein (APP) is proteolytically processed to release its NH(2)-terminal extracellular domain as a soluble APP (sAPP) that contains the inhibitor domain against gelatinase A. To investigate roles of sAPP in the regulation of gelatinase A activity, we examined the correlation between the activation of progelatinase A and processing of APP. We found that stimulation of HT1080 fibrosarcoma cells with concanavalin A led to an activation of endogenous progelatinase A and to a novel processing of APP, which releases a COOH-terminally truncated form of sAPP (sAPPtrc) into the culture medium. Reverse zymographic analysis showed that sAPPtrc lacked an inhibitory activity against gelatinase A. Analyses of production of sAPPtrc in the presence of various metalloproteinase inhibitors showed that membrane type 1 matrix metalloproteinase (MT1-MMP), an activator of progelatinase A, is most likely responsible for the production of sAPPtrc. When the concanavalin A-stimulated HT1080 cells were cultured in the condition that inhibited MT1-MMP activity, sAPP and APP were associated with the extracellular matrix deposited by the cells, whereas these gelatinase A inhibitors in the matrix were displaced by sAPPtrc after exertion of MT1-MMP activity. Taken together, these data support a model in which MT1-MMP-catalyzed release of sAPPtrc leads to reduction of the extracellular matrix-associated gelatinase A inhibitor, sAPP, thus making it feasible for gelatinase A to exert proteolytic activity only near its activator, MT1-MMP.

Amyloid beta-Protein Precursor↗

PubMed2000

Analysis of tissue inhibitor of metalloproteinases-2 effect on pro-matrix metalloproteinase-2 activation by membrane-type 1 matrix metalloproteinase using baculovirus/insect-cell expression system.

Membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14) is known to activate pro-matrix metalloproteinase-2 (pro-MMP-2; progelatinase A) on the cell surface. To analyse the tissue inhibitor of metalloproteinases-2 (TIMP-2) effect on activation of pro-MMP-2 by MT1-MMP, we have expressed the full-size MT1-MMP (fMT1-MMP) and a transmembrane (TM)-domain-deleted soluble MT1-MMP (sMT1-MMP) in the baculovirus/Sf9 (Spodoptera frugiperda 9) insect-cell system, where neither endogenous gelatinolytic MMPs nor TIMP-2 are expressed. Both fMT1-MMP and sMT1-MMP expressed in the expression system were found not to contain the pro-domain and were able to activate the TIMP-2-free pro-MMP-2. Both in the insect cells and in vitro, activation of pro-MMP-2 by fMT1-MMP was enhanced at low concentrations of TIMP-2 and inhibited by its higher concentrations. The maximal enhancing effect was detected at 0.05 molar fraction of TIMP-2/fMT1-MMP. In contrast, activation of pro-MMP-2 by sMT1-MMP was dose-dependently inhibited by TIMP-2. These results demonstrate that the TM domain of MT1-MMP is not required for the ability to activate pro-MMP-2, but is required for the enhancing effect of TIMP-2 on pro-MMP-2 activation by recruiting pro-MMP-2 to the MT1-MMP-TIMP-2 complex as a cell-surface pro-MMP-2 receptor. Moreover, our data strongly suggest that the pro-domain of MT1-MMP is not required for the TIMP-2-mediated enhancing effect on pro-MMP-2 activation. In addition, the pro-MMP-2 in the MT1-MMP-TIMP-2-pro-MMP-2 ternary complex was not activated without external activator, but readily by addition of sMT1-MMP. This result demonstrates that MT1-MMP free of TIMP-2 would be the enzyme responsible for activation of the pro-MMP-2 in the ternary complex under physiological conditions.

Animals↗

PubMed1998

Induction of matrix metalloproteinase activation cascades based on membrane-type 1 matrix metalloproteinase: associated activation of gelatinase A, gelatinase B and collagenase 3.

SW1353 chondrosarcoma cells cultured in the presence of interleukin-1, concanavalin A or PMA secreted procollagenase 3 (matrix metalloproteinase-13). The enzyme was detected in the culture medium by Western blotting using a specific polyclonal antibody raised against recombinant human procollagenase 3. Oncostatin M enhanced the interleukin-1-induced production of procollagenase 3, whereas interleukin-4 decreased procollagenase 3 synthesis. The enzyme was latent except when the cells had been treated with concanavalin A, when a processed form of 48 kDa, which corresponds to the active form, was found in the culture medium and collagenolytic activity was detected by degradation of 14C-labelled type I collagen. The concanavalin A-induced activation of procollagenase 3 coincided with the processing of progelatinase A (matrix metalloproteinase-2) by the cells, as measured by gelatin zymography. In addition, progelatinase B (matrix metalloproteinase-9) was activated when gelatinase A and collagenase 3 were in their active forms. Concanavalin A treatment of SW1353 cells increased the amount of membrane-type-1 matrix metalloproteinase protein in the cell membranes, suggesting that this membrane-bound enzyme participates in an activation cascade involving collagenase 3 and the gelatinases. This cascade was effectively inhibited by tissue inhibitors of metalloproteinases-2 and -3. Tissue inhibitor of metalloproteinases-1, which is a much weaker inhibitor of membrane-type 1 matrix metalloproteinase than tissue inhibitors of metalloproteinases-2 and -3 [Will, Atkinson, Butler, Smith and Murphy (1996) J. Biol. Chem. 271, 17119-17123], was a weaker inhibitor of the activation cascade.

Blotting, Western↗

PubMed1999

Ovarian carcinoma regulation of matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase through beta1 integrin.

Culturing DOV 13 ovarian carcinoma cells on three-dimensional collagen lattice but not on thin-layer collagen induces processing of promatrix metalloproteinase (MMP)-2 to a M(r) 62,000 form, suggesting that multivalent integrin aggregation may participate in proteinase regulation. To address the role of collagen-binding integrins in this event, we treated DOV 13 cells with soluble beta1 integrin antibodies (clones P4C10 or 21C8) or beta1 integrin antibodies immobilized on latex beads to promote integrin aggregation. Divalent ligation of beta1 integrins with soluble P4C10 antibodies stimulated expression of pro-MMP-2 and its inhibitor, tissue inhibitor of metalloproteinase-2, whereas soluble 21C8 antibodies had no effect. Aggregation of beta1 integrins with immobilized 21C8 or P4C10 antibodies stimulated MMP-dependent pro-MMP-2 activation and accumulation of a M(r) 43,000 form of membrane type 1 MMP (MT1-MMP), a cell surface activator of pro-MMP-2, in cell extracts. beta1 integrin-mediated MMP-2 activation required protein synthesis and tyrosine kinase signaling and was reduced by an inhibitor of gene transcription. Treatment of control cells with concanavalin A stimulated MMP-dependent pro-MMP-2 activation and accumulation of M(r) 55,000 and 43,000 forms of MT1-MMP in cell extracts. Addition of either the MMP inhibitor GM-6001-X or exogenous tissue inhibitor of metalloproteinase-2 to concanavalin A-treated cells resulted in loss of the M(r) 43,000 form of MT1-MMP and accumulation of the M(r) 55,000 form of the enzyme in cell extracts, suggesting that the M(r) 43,000 form is a product of MMP-dependent M(r) 55,000 MT1-MMP proteolysis. Together, these data suggest that beta1 integrin stimulation of pro-MMP-2 activation involves MT1-MMP posttranslational processing and requires multivalent integrin aggregation.

Collagen↗

PubMed1996

Membrane-type matrix metalloproteinase 1 is a gelatinolytic enzyme and is secreted in a complex with tissue inhibitor of metalloproteinases 2.

The processing mechanism and gelatinolytic activity of the membrane-type matrix metalloproteinase 1 (MT-MMP-1) were examined by expressing in COS-1 cells a deletion mutant of MT-MMP-1 lacking the trans-membrane domain (delta MT1) and its site-directed mutant with a furin-resistant sequence in the propeptide domain (mutant delta MT1). delta MT1, but not mutant delta MT1, was processed to an active form and exhibited gelatinolytic activity as seen using gelatin zymography. delta MT1 isolated in a complex form with tissue inhibitor of metalloproteinases 2 (TIMP-2) from the stable transfectants demonstrated the NH2-terminal sequence of Ala113-IIe-Gln-Leu, indicating cleavage at one amino acid down-stream from the furin recognition sequence. The delta MT1/TIMP-2 complex formed a ternary complex with proMMP-2 through the COOH termini of TIMP-2 and proMMP-2. A human breast carcinoma cell line (MDA-MB-231 cells) also secreted MT-MMP-1 into culture media, which was purified in a complex form with TIMP-2 and showed gelatinolytic activity as seen using zymography. These results demonstrate for the first time that MT-MMP-1 is a gelatinolytic enzyme and secreted from cells in a complex with TIMP-2, which can form a ternary complex of MT-MMP-1/TIMP2/proMMP-2.

Animals↗

PubMed2003

Interleukin 10 blocks matrix metalloproteinase-2 and membrane type 1-matrix metalloproteinase synthesis in primary human prostate tumor lines.

Insulin-like growth factor (IGF) I has been shown previously to up-regulate matrix metalloproteinase-2 (MMP-2) production, whereas the interleukin (IL) 10/IL-10 receptor axis has been found to down-regulate MMP-2 synthesis in tumor cells. In this paper, we showed that IL-10 activation of the IL-10 receptor blocked MMP-2 and membrane type 1 (MT1) -MMP transcription and protein synthesis in nonimmortalized primary human prostate cell strains (i.e., HPCA-10a and HPCA-10c) derived from high-grade cancer. Northern blots, Western blots, and ELISAs showed that IL-10 suppressed IGF-I induction of MMP-2 and MT1-MMP mRNA synthesis in these cell strains (P < 0.001). Inhibition studies with IL-10 and IGF-I receptor antibodies plus transfections experiments with IL-10 sense, and IGF-I receptor antisense constructs confirmed these results. Finally, transient transfection experiments and chloramphenicol acetyltransferase assays with different regions of the 5' promoter region of the MMP-2 gene (-1659 to -555 bp) additionally showed that IGF-I stimulated p53-dependent plasmid catecholamine acetyltransferase activity and that IL-10 blocked IGF-I-induced plasmid catecholamine acetyltransferase activity. Electrophoretic mobility shift assays revealed that IL-10 induced protein(s) binding to a putative "silencer element" (-1309 to -555 fragment) downstream of the p53 binding site (-1649 to -1640). The data show that IL-10 blocks IGF-I activation of MMP-2 and MT1-MMP mRNA expression and protein synthesis in primary prostate cell strains.

Binding Sites↗

PubMed2004

Increased matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase activity and expression in heterotopically transplanted murine tracheas.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is the most common long-term cause of morbidity and mortality after heart-lung or lung transplantation. One pathologic feature of BOS is infiltration of fibroblasts and connective tissue products into the airway lumen, which form a fibrous, collagen-rich occlusion. Heterotopically transplanted allogeneic murine tracheal stenosis resemble BOS in the development of obliterans airway disease. Matrix metalloproteinases (MMPs) are key enzymes involved in tissue remodeling and, clinically, have several roles in pulmonary diseases. Among the MMP family, type IV collagenases, MMP-2 and MMP-9, have high gelatinolytic activity and are thought to play a role in several pulmonary diseases. Membrane type 1 MMP (MT1-MMP) activates the zymogen of MMP-2 (proMMP-2, 72 kd), and activated MMP-2 (active MMP-2, 62 kd) degrades type IV collagen and plays an important role in clinical pulmonary disease. In this study, we examine the expression of MMP-2, its activator MT1-MMP and MMP-9 in BOS using murine trachea transplantation models. METHODS: Rats were divided into 5 experimental groups (n = 10 in each group). Group I was a control group with intact tracheas. Animals with tracheal grafts underwent heterotopically syngeneic (Groups II and III) or allogeneic (Groups IV and V) transplantation. The recipient rats were killed 7 days (Groups II and IV) or 28 days (Groups III and V) after transplantation. The harvested tracheal grafts were examined histologically. MMP activity was assessed using gelatin zymography analysis, and MMP-2 and MT1-MMP gene expression was examined by quantitative real-time polymerase chain reaction analysis. Distribution of gelatinolytic activity was studied using in situ zymography. RESULTS: There was little histologic change in the intact trachea (Group I) and in all isografts (Groups II and III). Fibrotic tissues in Group V significantly occluded the tracheal lumen, and there was severe lymphocyte infiltration in Group IV. According to gelatin zymography, proMMP-9 was faint at 7 days, but activated MMP-9 was not present in all groups. The MMP-2 gelatinolytic bands were predominant; the activation in Group V was significantly greater than that in Group IV, and in Group III it was significantly greater than that in Group II. Gene expression of both MMP-2 and MT1-MMP were significantly higher in Group V than in the other groups (p < 0.01), and MMP-2 was clearly activated. Gelatinolytic activity was localized in the fibrotic tissues or lymphocytes of thickening lumen after destruction of the epithelium by stenosis. CONCLUSIONS: These results demonstrate that MMP-2, together with its activator MT1-MMP, may have an important role in the development of BOS, which is associated with destruction of the tracheal epithelium, leading to fibrosis.

Animals↗

PubMed2003

Tetraspanin CD63 promotes targeting and lysosomal proteolysis of membrane-type 1 matrix metalloproteinase.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is known to be internalized from cell surface, however, the fate of internalized MT1-MMP is still unknown. Here we demonstrate that at least a part of internalized MT1-MMP is targeted for lysosomal proteolysis. Treatment with an inhibitor of lysosomal proteinases chloroquine suppressed degradation of internalized MT1-MMP and induced accumulation of MT1-MMP in CD63-positive lysosomes. Ectopic expression of CD63 accelerated degradation of MT1-MMP, which was blocked by chloroquine. MT1-MMP, and CD63 were shown to form a complex through hemopexin-like domain of MT1-MMP and N-terminal region of CD63, and thus accelerated degradation of MT1-MMP was not observed with mutants lacking these domains. CD63 mutant lacking lysosomal targeting motif was unable to promote MT1-MMP degradation. These results suggest that CD63 regulates MT1-MMP by targeting to lysosomes.

Animals↗

PubMed2005

CD44 binding through the hemopexin-like domain is critical for its shedding by membrane-type 1 matrix metalloproteinase.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a potent modulator of pericellular environment through its proteolytic activity and promotes migration, invasion, and proliferation of tumor cells. During cell migration, MT1-MMP binds to CD44H, a major hyaluronan receptor, through the hemopexin-like (HPX) domain and localizes at the migration front. MT1-MMP is also responsible for shedding CD44H, which supports CD44H-mediated cell migration. In this study, we asked whether the binding of MT1-MMP to CD44H is a prerequisite step for the successive shedding. Deletion of the HPX domain deprived MT1-MMP of its shedding activity. Furthermore, disruption of the CD44H/MT1-MMP complex by overexpressing the HPX fragments resulted in inhibition of the shedding. Thus, the CD44H in the complex appears to be the direct substrate of MT1-MMP for shedding. Interestingly, other members of the MT-MMP family showed varied extents of CD44H shedding. Domain swapping between MT1-MMP and other MT-MMPs revealed that the ability of the HPX domains to bind CD44H is conserved among them. However, the shedding activity was different depending on the catalytic domains. The conserved binding ability of the HPX domains suggests that CD44H may act as a core molecule assembling multiple MT-MMPs on the cell surface.

Binding Sites↗