The complete structure of the human gene for 92-kDa type IV collagenase was determined. Two overlapping genomic clones spanning
26 kilobases (kb) of genomic DNA were shown to contain the entire ...7.7-kb structural gene together with 15 and 3.5 kb of 5'-end
and 3'-end flanking regions, respectively. The 92-kDa type IV collagenase gene contains 13 exons as does the 72-kDa type IV
collagenase gene. All intron locations of the 92-kDa enzyme gene coincided with intron locations in the 72-kDa enzyme gene.
Exons 5, 6, and 7 which were 174, 174, and 177 base pairs long, respectively, each encoded one complete internal repeat which
resembles the collagen-binding domains of fibronectin. The sequence coding for a unique 48-residue segment in the 92-kDa type
IV collagenase that has no counterpart in other metalloproteinases was not present in a separate exon, but was contained in
exon 9 which also codes for sequences with homology to the other metalloproteinases. The initiation site for transcription
was determined by primer extension analysis. Sequencing analysis of 599 base pairs of the 5'-end flanking region showed that
the promoter does not have a TATA motif, but a TTAAA sequence at position -29 to -25. A CAAT motif was not observed but there
was one GC box. Two putative 12-O-tetradecanoyl-phorbol-13-acetate (TPA) response elements, that might serve as binding sites
for the transcription factor AP-1 and a consensus sequence of a transforming growth factor beta 1 (TGF-beta 1) inhibitory
element were found in the promoter region. Gelatinase assay of enzyme secreted by cultured human fibrosarcoma cells (HT-1080)
revealed only low levels of 92-kDa type IV collagenase activity, whereas considerable activity of the 72-kDa enzyme was present.
Northern hybridization analysis confirmed these findings. Treatment of the HT-1080 cells with TPA resulted in induction of
the secretion of 92-kDa type IV collagenase activity. This induction could not be significantly inhibited by concomitant incubation
with TGF-beta 1. TPA and TGF-beta 1 did not markedly affect the activities of the 72-kDa enzyme. The activities of the secreted
92- and 72-kDa enzymes by HT-1080 cells correlated with the amounts of mRNA as estimated by Northern analyses.
Regulation of the activity of proteolytic enzymes is of major importance in the turnover of connective tissues. The search for physiologically relevant activation mechanisms of principal ...tissue-degrading enzymes, e.g., metalloproteinases, has therefore been of wide interest. We have now studied whether the initiating factor of the fibrinolytic system, urokinase plasminogen activator (u-PA), may also function in the early steps of activation of one of the metalloproteinases, the M(r) 72,000 gelatinase/type IV collagenase produced by cultured fibroblasts. Treatment of the secreted M(r) 72,000 proteinase by u-PA yielded a cleavage product of M(r) 62,000 as revealed by fluorography of radioactively labeled proteins as well as by gelatin zymography SDS-PAGE gels. The u-PA-catalyzed cleavage of the M(r) 72,000 proteinase was blocked by anti-u-PA antibodies, but was unaffected by the plasmin inhibitor aprotinin, thus indicating a specific action for the activator. On the contrary, the tissue activator of plasminogen, t-PA, did not cleave the type IV collagenase in similar assays. u-PA-catalyzed cleavage of recombinant type IV collagenase, produced in a baculovirus expression system, yielded a similar M(r) 62,000 activity in gelatinolysis assay. Zymograms of the isolated pericellular matrices of cultured fibroblasts also revealed M(r) 72,000 gelatinolytic polypeptide that was converted to an M(r) 62,000 form by u-PA. Both polypeptides were recognized in immunoblotting by antibodies against the gelatinase/type IV collagenase, suggesting immunological identity with the secreted enzyme. Thus the M(r) 72,000 gelatinase/type IV collagenase is not only secreted, but also deposited into the pericellular fibroblast matrix, and both forms are substrates for u-PA. The results suggest a new potential role for u-PA as a direct regulator of metalloproteinase-mediated extracellular proteolysis via the cleavage of the M(r) 72,000 gelatinase/type IV collagenase to an M(r) 62,000 form.
Liver cells are considered the principal source of plasma vitronectin. The human hepatoma cell line HepG2 produces vitronectin into its culture medium. In the current work we have analyzed the ...regulation of vitronectin by transforming growth factor‐β1 (TGFβ1) in this hepatoma cell line by Northern hybridization, polypeptide and immunoprecipitation analyses and compared the response to another TGFβ‐regulated gene, plasminogen activator inhibitor (PAI‐1). Rabbit antibodies raised against human plasma‐derived vitronectin were used in immunodetection. Polypeptide and immunoprecipitation analyses of the medium and cells, as well as immunoblotting analysis of the cells and their extracellular matrices, indicated enhanced TGFβ1‐induced production and extracellular deposition of vitronectin. Accordingly, TGFβ1 enhanced the expression of vitronectin mRNA at picomolar concentrations (2–20 ng/ml) as shown by Northern hybridization analysis. Comparison of the temporal TGFβ induction profiles of vitronectin and PAI‐1 mRNAs showed that vitronectin was induced more slowly but the vitronectin mRNAs persisted longer. In addition, platelet‐derived and epidermal growth factors had an effect on vitronectin expression, but it was of lower magnitude. TGFβ1 enhanced the expression of PAI‐1 but, unlike previous reports, epidermal growth factor did not have any notable effect on PAI‐1 in these cells. The results indicate that TGFβ1 is an efficient regulator of the production of vitronectin by HepG2 cells and that PAI‐1 and vitronectin are not coordinately regulated. In addition, with affinity purified antibodies to vitronectin receptor, we observed strong enhancement of the α subunit of the receptor in response to TGFβ1. These effects of TGFβ are probably involved in various processes of the liver where matrix induction and controlled pericellular proteolysis is needed, as in tissue repair.
Overexpression of membrane‐type matrix metalloproteinase (MT‐MMP‐1) results in the activation of both endogenous and exogenous 72‐kDa gelatinase. To understand the effects of MT‐MMP‐1 on 72‐kDa ...gelatinase activation, we analyzed its expression in human fibroblasts and HT‐1080 fibrosarcoma cells. Both cell types expressed the MT‐MMP‐1 mRNA constitutively at a considerable level and treatment of cells with PMA enhanced the expression about 2–3‐fold. Concanavalin A treatment increased MT‐MMP‐1 mRNA levels in fibroblasts about 4‐fold. Induction of MT‐MMP‐1 by phorbol 12‐myristate 13‐acetate (PMA) required protein synthesis as shown by cycloheximide inhibition. The induction was also inhibited by dexamethasone. Analysis of MT‐MMP‐1 mRNA stability using actinomycin D indicated that the half‐life was rather long and not affected by PMA, suggesting transcriptional regulation. Only HT‐1080 cells had significant 72‐kDa gelatinase processing activity after treatment with PMA or concanavalin A, while fibroblasts were virtually negative. Immunoblotting analysis of fibroblast lysates indicated that MT‐MMP‐1 was present mainly in a 60‐kDa form. PMA and concanavalin A caused 2–4‐fold increases in its protein levels, while in HT‐1080 cells PMA, concanavalin A, or overexpression of MT‐MMP‐1 did not significantly enhance the level of the 60‐kDa protein. Instead, an immunoreactive, proteolytically processed 43‐kDa form was observed, and its appearance correlated to 72‐kDa gelatinase processing activity. Thus 72‐kDa gelatinase activation, while enhanced by MT‐MMP‐1 expression, needs additional co‐operating factors.
The role of matrix metalloproteinases (MMP), especially newly described MMP, in trophoblast invasion during human embryo implantation is poorly understood. In this report, using a model of early ...pregnancy in the rhesus monkey, we have examined the expression and localization of the most recently identified MMP, MMP‐28/epilysin, transcript and protein in macaque uterine samples on days 12, 18 and 26 of pregnancy. MMP‐28 mRNA expression was shown by in‐situ hybridization after day 12 of pregnancy, and both the syncytial and the cytotrophoblastic cell layers of placental villi, the cytotrophoblast cells of the trophoblastic column, and the extravillous trophoblast cells of trophoblastic shell were primary producers of MMP‐28 transcript. Expression of MMP‐28 mRNA was undetectable in the endovascular trophoblast cells, decidual cells, luminal and glandular epithelium, arterioles, and myometrium. RT–PCR analysis amplified a fragment of 258 nucleotides from rhesus monkey uterine samples containing implantation sites on days 18 and 26. The cDNA fragment, following sequencing, was confirmed to be part of the haemopexin‐like domain of MMP‐28. It has 95% identity with the corresponding region of human MMP‐28 gene. Immunohistochemical analysis further demonstrated that the localization of MMP‐28 protein was similar to that of its mRNA. The restricted distribution pattern of this novel MMP in the villous and extravillous trophoblasts during rhesus monkey early pregnancy suggests a potential role in trophoblast invasion associated with embryo implantation.
Human matrix metalloproteinase-21 (MMP-21), the newest member of the MMP gene family, has been suggested to play an important role in embryogenesis and tumor progression and to be a target of the ...Wnt, Pax, and Notch signaling pathways. Here we report detection of MMP-21 by RT-PCR in mouse embryos aged 10.5, 12.5, 13.5, and 16.5 days, as well as in various adult murine organs. In both humans and mice, MMP-21 protein was detected in the epithelial cells of developing kidney, intestine, neuroectoderm, and skin but not in normal adult skin using immunohistochemistry with two unrelated antibodies. However, it was present in invasive cancer cells of aggressive subtypes of basal and squamous cell carcinomas, although it was not expressed in skin disorders characterized by mere keratinocyte hyperproliferation. Of several cytokines tested, transforming growth factor-beta1 induced MMP-21 in vitro in HaCaTs and keratinocytes as judged by real-time quantitative TaqMan PCR. Although suprabasal differentiating keratinocytes expressed MMP-21 in developing skin in vivo, MMP-21-positive keratinocytes were detected by immunohistochemistry in both low and high calcium cultures. MMP-21 expression was not up-regulated by ras transformation in HaCaT cell lines (HaCaT, A5, II-4, and RT3); in skin and colon cancers, its expression did not associate with apoptosis, beta-catenin transactivation, or epithelial MMPs-9 and -10. However, MMP-21 protein was found in the same regions as MMP-7 but not in the same cells. Our results suggest that during development, MMP-21 expression is temporally and spatially tightly controlled. Unlike many classical MMPs, it is present in various normal adult tissues. Among epithelial MMPs, MMP-21 has a unique expression pattern in cancer.
Human matrix metalloproteinase-21 (MMP-21), the newest member of the MMP gene family, has been suggested to play an important role in embryogenesis and tumor progression and to be a target of the ...Wnt, Pax, and Notch signaling pathways. Here we report detection of MMP-21 by RT-PCR in mouse embryos aged 10.5, 12.5, 13.5, and 16.5 days, as well as in various adult murine organs. In both humans and mice, MMP-21 protein was detected in the epithelial cells of developing kidney, intestine, neuroectoderm, and skin but not in normal adult skin using immunohistochemistry with two unrelated antibodies. However, it was present in invasive cancer cells of aggressive subtypes of basal and squamous cell carcinomas, although it was not expressed in skin disorders characterized by mere keratinocyte hyperproliferation. Of several cytokines tested, transforming growth factor-beta1 induced MMP-21 in vitro in HaCaTs and keratinocytes as judged by real-time quantitative TaqMan PCR. Although suprabasal differentiating keratinocytes expressed MMP-21 in developing skin in vivo, MMP-21-positive keratinocytes were detected by immunohistochemistry in both low and high calcium cultures. MMP-21 expression was not up-regulated by ras transformation in HaCaT cell lines (HaCaT, A5, II-4, and RT3); in skin and colon cancers, its expression did not associate with apoptosis, beta-catenin transactivation, or epithelial MMPs-9 and -10. However, MMP-21 protein was found in the same regions as MMP-7 but not in the same cells. Our results suggest that during development, MMP-21 expression is temporally and spatially tightly controlled. Unlike many classical MMPs, it is present in various normal adult tissues. Among epithelial MMPs, MMP-21 has a unique expression pattern in cancer.
Cyclosporin A is successfully used in the treatment of scleroderma, a condition with excessive deposition of collagen in the dermis. Cultured human dermal fibroblasts were used as a model to study ...the effects of cyclosporin A on metalloproteinase expression and activity. Fibroblasts were treated with collagenase inducing agents, phorbol 12-myristate 13-acetate (PMA), cytokines interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNFα), and the calcium ionophore A23187 in the presence of cyclosporin A under serum-free conditions, and alterations in metalloproteinase expression were studied by Northern hybridization and immunoblotting analyses, and assays for collagenolytic activity. Induction of collagenase expression by PMA and cytokines was enhanced severalfold by 1-10 μM cyclosporin A. Treatment of cells with cyclosporin A alone caused only a minor increase in collagenase mRNA levels. The secretion of immunoreactive collagenase protein and the level of p-aminophenylmercuric acetate activatable collagenase activity were increased by PMA and further enhanced by cyclosporin A. The expression of the other metalloproteinases stromelysin-1, 92-kD gelatinase, and 72-kD gelatinase or metalloproteinase inhibitor TIMP-1 were not affected by cyclosporin A. Time dependence analysis of the expression of the mRNAs for c-jun and junB indicated that the induction of these genes persisted significantly longer in cells treated with both PMA and cyclosporin A than in cells treated with PMA alone. Enhanced induction of collagenase mRNA may thus result from prolonged AP-1 activity. The results indicate that cyclosporin A potently enhances the expression of collagenase in dermal fibroblasts.
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