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Nicola C Partridge - One of the best experts on this subject based on the ideXlab platform.

  • smad3 interacts with junb and cbfa1 runx2 for transforming growth factor β1 stimulated Collagenase 3 expression in human breast cancer cells
    Journal of Biological Chemistry, 2004
    Co-Authors: N Selvamurugan, Sukyee Kwok, Nicola C Partridge
    Abstract:

    We have previously shown that transforming growth factor (TGF)-β1, a crucial molecule in metastatic bone cancer, stimulates Collagenase-3 expression in the human breast cancer cell line, MDA-MB231. To understand the molecular mechanisms responsible for TGF-β1 response on Collagenase-3 promoter activity, a functional analysis of the promoter region of the Collagenase-3 gene was carried out, and we identified the distal runt domain (RD) and proximal RD/activator protein-1 (AP-1) sites as necessary for full TGF-β1-stimulated Collagenase-3 promoter activity. Gel shift, real time reverse transcriptase-PCR, and Western blot analyses showed increased levels of c-Jun, JunB, and Cbfa1/Runx2 upon TGF-β1 treatment in MDA-MB231 cells. Co-immunoprecipitation in vitro studies identified no physical interaction between JunB and Cbfa1/Runx2, whereas Smad3 interacted with both. Chromatin immunoprecipitation experiments confirmed interaction of Smad3 with JunB and Cbfa1/Runx2. Under basal conditions, Cbfa1/Runx2 bound to both the proximal RD/AP-1 and distal RD sites. In response to TGF-β1, Cbfa1/Runx2 was seen only at the distal RD site, whereas JunB occupied the proximal RD/AP-1 site. An assemblage of Smad3, JunB, and Cbfa1/Runx2 at the distal RD site of the Collagenase-3 promoter occurred in response to TGF-β1 in MDA-MB231 cells. Co-transfection of Smad3, JunB, and Cbfa1/Runx2 constructs along with a constitutively active TGF-β type I receptor construct identified functional interaction of these proteins and transcriptional activation of the Collagenase-3 gene by TGF-β1. Taken together, our results suggest that TGF-β1 stimulated JunB and Cbfa1/Runx2 to bind to their respective DNA consensus sites and that Smad3 is likely to stabilize their interaction to confer functional TGF-β1-stimulation of Collagenase-3 expression in MDA-MB231 cells.

  • Smad3 Interacts with JunB and Cbfa1/Runx2 for Transforming Growth Factor-β1-stimulated Collagenase-3 Expression in Human Breast Cancer Cells
    The Journal of biological chemistry, 2004
    Co-Authors: Nagarajan Selvamurugan, Sukyee Kwok, Nicola C Partridge
    Abstract:

    Abstract We have previously shown that transforming growth factor (TGF)-β1, a crucial molecule in metastatic bone cancer, stimulates Collagenase-3 expression in the human breast cancer cell line, MDA-MB231. To understand the molecular mechanisms responsible for TGF-β1 response on Collagenase-3 promoter activity, a functional analysis of the promoter region of the Collagenase-3 gene was carried out, and we identified the distal runt domain (RD) and proximal RD/activator protein-1 (AP-1) sites as necessary for full TGF-β1-stimulated Collagenase-3 promoter activity. Gel shift, real time reverse transcriptase-PCR, and Western blot analyses showed increased levels of c-Jun, JunB, and Cbfa1/Runx2 upon TGF-β1 treatment in MDA-MB231 cells. Co-immunoprecipitation in vitro studies identified no physical interaction between JunB and Cbfa1/Runx2, whereas Smad3 interacted with both. Chromatin immunoprecipitation experiments confirmed interaction of Smad3 with JunB and Cbfa1/Runx2. Under basal conditions, Cbfa1/Runx2 bound to both the proximal RD/AP-1 and distal RD sites. In response to TGF-β1, Cbfa1/Runx2 was seen only at the distal RD site, whereas JunB occupied the proximal RD/AP-1 site. An assemblage of Smad3, JunB, and Cbfa1/Runx2 at the distal RD site of the Collagenase-3 promoter occurred in response to TGF-β1 in MDA-MB231 cells. Co-transfection of Smad3, JunB, and Cbfa1/Runx2 constructs along with a constitutively active TGF-β type I receptor construct identified functional interaction of these proteins and transcriptional activation of the Collagenase-3 gene by TGF-β1. Taken together, our results suggest that TGF-β1 stimulated JunB and Cbfa1/Runx2 to bind to their respective DNA consensus sites and that Smad3 is likely to stabilize their interaction to confer functional TGF-β1-stimulation of Collagenase-3 expression in MDA-MB231 cells.

  • Transforming Growth Factor-β1 Regulation of Collagenase-3 Expression in Osteoblastic Cells by Cross-talk between the Smad and MAPK Signaling Pathways and Their Components, Smad2 and Runx2
    The Journal of biological chemistry, 2004
    Co-Authors: Nagarajan Selvamurugan, Sukyee Kwok, Tamara Alliston, Michael Reiss, Nicola C Partridge
    Abstract:

    Abstract Transforming growth factor-β (TGF-β) plays a key role in osteoblast differentiation and bone development and remodeling. Collagenase-3 (matrix metalloproteinase-13) is expressed by osteoblasts and seems to be involved in osteoclastic bone resorption. Here, we show that TGF-β1 stimulates Collagenase-3 expression in the rat osteoblastic cell line UMR 106-01 and requires de novo protein synthesis. Dominant-negative Smad2/3 constructs indicated that Smad signaling is essential for TGF-β1-stimulated Collagenase-3 promoter activity. Inhibitors of the ERK1/2 and p38 MAPK pathways, but not the JNK pathway, reduced TGF-β1-stimulated Collagenase-3 expression, indicating that the p38 MAPK and ERK1/2 pathways are also required for TGF-β1-stimulated Collagenase-3 expression in UMR 106-01 cells. These inhibitors did not prevent nuclear localization of Smad proteins, but they inhibited Smad-mediated transcriptional activation. We have shown for the first time that Runx2 (a bone transcription factor and a potential substrate for the MAPK pathway) is phosphorylated in response to TGF-β1 treatment in osteoblastic cells. Cotransfection of Smad2 and Runx2 constructs had a cooperative effect on TGF-β1-stimulated Collagenase-3 promoter activity in these cells. We further identified ligand-independent physical interaction between Smad2 and Runx2. Taken together, our results provide an important role for cross-talk between the Smad and MAPK pathways and their components in expression of Collagenase-3 following TGF-β1 treatment in UMR 106-01 cells.

  • Transcriptional activation of Collagenase‐3 by transforming growth factor‐β1 is via MAPK and Smad pathways in human breast cancer cells
    FEBS Letters, 2002
    Co-Authors: Nagarajan Selvamurugan, Ziawei Fung, Nicola C Partridge
    Abstract:

    Transforming growth factor (TGF)-β1, a crucial molecule in metastatic bone cancer, stimulates Collagenase-3 expression in the human breast cancer cell line, MDA-MB231. Cycloheximide inhibited this stimulation, indicating that de novo protein synthesis was essential for this response. We examined whether mitogen-activated protein kinase (MAPK) and/or Smad pathways are involved in TGF-β1-stimulated Collagenase-3 expression in MDA-MB231 cells. Biochemical blockade of extracellular regulated kinase-1/2 and p38 MAPK pathways partially abolished TGF-β1-stimulated Collagenase-3 mRNA expression; whereas overexpression of a dominant negative form of Smad3 completely blocked the TGF-β1-response. These data indicate that TGF-β1-induced MAPK and Smad pathways are involved in TGF-β1-stimulated Collagenase-3 expression in MDA-MB231 cells.

  • Transcriptional activation of Collagenase-3 by transforming growth factor-beta1 is via MAPK and Smad pathways in human breast cancer cells.
    FEBS letters, 2002
    Co-Authors: Nagarajan Selvamurugan, Ziawei Fung, Nicola C Partridge
    Abstract:

    Transforming growth factor (TGF)-beta1, a crucial molecule in metastatic bone cancer, stimulates Collagenase-3 expression in the human breast cancer cell line, MDA-MB231. Cycloheximide inhibited this stimulation, indicating that de novo protein synthesis was essential for this response. We examined whether mitogen-activated protein kinase (MAPK) and/or Smad pathways are involved in TGF-beta1-stimulated Collagenase-3 expression in MDA-MB231 cells. Biochemical blockade of extracellular regulated kinase-1/2 and p38 MAPK pathways partially abolished TGF-beta1-stimulated Collagenase-3 mRNA expression; whereas overexpression of a dominant negative form of Smad3 completely blocked the TGF-beta1-response. These data indicate that TGF-beta1-induced MAPK and Smad pathways are involved in TGF-beta1-stimulated Collagenase-3 expression in MDA-MB231 cells.

Carlos López-otín - One of the best experts on this subject based on the ideXlab platform.

  • Critical roles for Collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification.
    Proceedings of the National Academy of Sciences of the United States of America, 2004
    Co-Authors: Masaki Inada, Carlos López-otín, Yingmin Wang, Michael H. Byrne, Mahboob U. Rahman, Chisato Miyaura, Stephen M. Krane
    Abstract:

    Collagenase-3 (MMP13), a member of the matrix metalloproteinase (MMP) family of neutral endopeptidases, is expressed in the skeleton during embryonic development and is highly overexpressed in human carcinomas and in chondrocytes and synovial cells in rheumatoid arthritis and osteoarthritis. To determine the functional roles of Mmp13, we generated Mmp13-null mice that showed profound defects in growth plate cartilage with markedly increased hypertrophic domains as well as delay in endochondral ossification and formation and vascularization of primary ossification centers. Absence of Mmp13 resulted in significant interstitial collagen accumulation due, in part, to the lack of appropriate Collagenase-mediated cleavage that normally occurs in growth plates and primary ossification centers. Cartilaginous growth plate abnormalities persisted in adult mice and phenocopied defects observed in human hereditary chondrodysplasias. Our findings demonstrate a unique role of Mmp13 in skeletal development.

  • An overview of Collagenase-3 expression in malignant tumors and analysis of its potential value as a target in antitumor therapies
    Clinica chimica acta; international journal of clinical chemistry, 2000
    Co-Authors: Alberto M. Pendás, José M. P. Freije, Milagros Balbín, José A. Uría, Maria G. Jiménez, Carlos López-otín
    Abstract:

    Collagenase-3 (MMP-13) is a member of the matrix metalloproteinase family of endopeptidases that is characterized by a potent degrading activity against a wide spectrum of substrates. This enzyme was first detected in breast carcinomas but it is also overexpressed in a variety of malignant tumors including head and neck carcinomas, chondrosarcomas, skin carcinomas, and carcinomas of the female genital tract. Clinical studies have revealed that in all these tumors Collagenase-3 expression is associated with invasive and metastatic tumors. Analysis of the molecular mechanisms underlying its marked overexpression in malignant tumors has allowed to identify different cytokines, growth factors and tumor promoters with ability to up-regulate Collagenase-3 expression in tumor cells, or in stromal fibroblasts surrounding epithelial tumor cells. The first strategies designed to target this enzyme are being developed, and are mainly directed to prepare synthetic inhibitors with ability to selectively block the Collagenase-3 proteolytic activity. Alternatively, inhibitors of the signal transduction pathways mediating the expression of this enzyme by tumor cells may also be useful for Collagenase-3 targeting. These studies together with those performed on other enzymes associated with tumor processes may lead to the development of novel therapeutic strategies to control the progression and metastatic capacity of neoplastic cells.

  • Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization.
    The Journal of biological chemistry, 1999
    Co-Authors: O. Y. Barmina, Carlos López-otín, Hobart W. Walling, Gerald J. Fiacco, José M. P. Freije, John J. Jeffrey, Nicola C Partridge
    Abstract:

    We have previously identified a specific receptor for Collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that Collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of Collagenase-3 binding and internalization involving both a specific Collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-Collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific Collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize Collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize Collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of Collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the Collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

  • Collagenase 3 is a target of Cbfa1, a transcription factor of the runt gene family involved in bone formation.
    Molecular and cellular biology, 1999
    Co-Authors: Maria G. Jiménez, Milagros Balbín, José María Cuenca López, Jesus Alvarez, Toshihisa Komori, Carlos López-otín
    Abstract:

    The human matrix metalloproteinases (MMPs) or matrixins are a family of structurally related neutral proteinases that are collectively capable of degrading essentially all extracellular matrix components (9). These enzymes play a major role in normal tissue-remodeling processes such as embryonic development, ovulation, and wound healing (44, 81). In addition, abnormal expression of these proteases may contribute to a variety of pathological conditions characterized by matrix destruction, including rheumatoid arthritis (52), atherosclerosis (25), and cancer invasion and metastasis (43, 72). Recently, and based on the hypothesis that samples of human tumor specimens could be an appropriate material to identify novel proteinases potentially involved in the spread of cancer, we have cloned from a breast carcinoma cDNA library a new member of the MMP family of enzymes that has been called Collagenase 3 (MMP-13) (21, 55). Biochemical characterization of this enzyme has revealed that it degrades very efficiently the native helix of fibrillar collagens, with preferential activity on type II collagen. In addition, Collagenase 3 may also act as a potent gelatinase, thus contributing to further degrade the initial cleavage products of collagenolysis to small fragments suitable for subsequent metabolism (33). Furthermore, recent studies have shown that Collagenase 3 is also able to degrade the large cartilage proteoglycan aggrecan and other components of the extracellular matrix and basement membranes, including type IV collagen (19, 33, 35). Analysis of the expression of Collagenase 3 in human tissues has revealed that in addition to its presence in diverse malignant tumors including breast carcinomas (21, 26), chondrosarcomas (77), basal cell carcinomas of the skin (1), and head and neck carcinomas (13, 29), this enzyme is produced during fetal ossification (30, 70) and in destructive joint diseases such as osteoarthritis and rheumatoid arthritis (41, 49, 59). Recent studies have provided information on the mechanisms controlling human Collagenase 3 expression in pathological conditions. Thus, we have reported that this gene is predominantly expressed in fibroblasts adjacent to invasive breast cancer cells, in response to diffusible factors released from the epithelial tumor cells (76). A search of molecular factors with ability to induce Collagenase 3 expression in human fibroblasts has shown that interleukin-1 (IL-1), tetradecanoyl phorbol acetate (TPA), and transforming growth factor β (TGF-β) are able to up-regulate the expression of this gene (76, 78). Functional analysis of the Collagenase 3 gene promoter region has revealed that the inductive effects of all of these factors on the expression of Collagenase 3 are mediated in part by an AP-1 site present in the 5′-flanking region of this gene (56, 78). Similar studies using human chondrosarcoma cells have indicated that basic fibroblast growth factor (bFGF) may be a major in vivo modulator of Collagenase 3 expression in these malignant tumors (77). Furthermore, different groups have reported that IL-1β and tumor necrosis factor alpha (TNF-α) may induce Collagenase 3 expression in osteoarthritic cartilage (11, 59). However, in marked contrast to these data on human Collagenase 3 expression in pathological conditions, very little information is available on the mechanisms mediating its expression in normal conditions and, more specifically, in the process of bone formation, in which high levels of Collagenase 3 have been detected. Recent structural analysis of the 5′-flanking region of the human Collagenase 3 gene (56) has shown that it contains a sequence motif located at positions −133 to −139 that exhibits striking similarity to a sequence motif called nuclear matrix protein 2 (NMP-2) binding site (8, 47) or osteoblast-specific element 2 (OSE2) (15, 17). This sequence, originally described as a structural element essential for the osteoblastic expression of osteocalcin, is recognized by a transcription factor of the runt domain gene family, called Cbfa1 or Osf2 (7, 15, 17, 69, 83), that plays a major role in the expression of different osteoblast-specific genes (6, 7, 17, 37, 53). In this work we have evaluated the possibility that Cbfa1 is involved in the expression of Collagenase 3 during bone formation. It was recently reported that parathyroid hormone (PTH) regulates the rat Collagenase 3 promoter in osteoblastic cells through the cooperative interaction of an AP-1 site and a runt domain binding sequence recognized by runt domain proteins including Cbfa1 (67). Here, we provide in vitro and in vivo evidence that Collagenase 3 is a target of Cbfa1 in osteoblastic and chondrocytic cells. In addition, on the basis of these transcriptional regulation studies, together with the potent proteolytic activity of Collagenase 3 on bone and cartilage collagens, we propose that this enzyme may play a key role during fetal ossification.

  • Expression and regulation of Collagenase‐3 (MMP‐13) in human malignant tumors
    APMIS : acta pathologica microbiologica et immunologica Scandinavica, 1999
    Co-Authors: Milagros Balbín, Alberto M. Pendás, José M. P. Freije, José A. Uría, Maria G. Jiménez, Carlos López-otín
    Abstract:

    Human Collagenase-3 (MMP-13) is a matrix metalloproteinase originally identified in breast carcinomas. Recent studies have revealed that this enzyme is also produced by a variety of malignant tumors including head and neck carcinomas, chondrosarcomas and basal cell carcinomas of the skin. In all cases, the expression of Collagenase-3 is associated with aggressive tumors. Different cytokines, growth factors and tumor promoters are able to up-regulate Collagenase-3 expression in tumor cells or in stromal cells surrounding epithelial tumor cells. Functional analysis of the Collagenase-3 gene promoter has allowed the identification of AP-1 and OSE-2 elements mediating, at least in part, its expression in both normal and pathological conditions.

Johanne Martel-pelletier - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of Collagenase-3 (MMP-13) gene and protein expression in human cartilage: when simple things get complicated
    Arthritis Research & Therapy, 2003
    Co-Authors: Johanne Martel-pelletier, Ginette Tardif, J.-p. Pelletier
    Abstract:

    In cartilage, collagen type II is of particular importance as its breakdown results in the irreversible loss of structural integrity of the tissue. Within the protease, the Collagenases have a major involvement in this collagen network degradation. Evidence demonstrates that Collagenase-3 is the major enzyme accounting for collagen degradation in osteoarthritic (OA) cartilage. Collagenase-3 has a greater effect (five to 10 times) on type II collagen than Collagenase-1, and, in OA, is localized predominantly in the lower intermediate and deep layers of the cartilage, where type II collagen fibers are of the largest size and chondrocytes possess the most efficient capacity to reconstitute the extracellular matrix. Collagenase is upregulated in OA cartilage and is suggested to be implicated in cartilage remodeling in pathological conditions. Various factors induce its transcription, including proinflammatory cytokines and growth factors such as IL-1β, IL-17, tumor necrosis factor alpha, transforming growth factor beta and hepatocyte growth factor. Interestingly, we recently reported that transforming growth factor beta, but not IL-1β, treatment of normal cartilage mimicked the in situ Collagenase-3 distribution in OA cartilage. The proximal promoter sequence contains a TATA box, as well as AP-1, Ets/PEA-3, and OSE-2 binding sites. The AP-1 site is essential for both basal and proinflammatory cytokine inducible transcription, and the PEA-3 site exerts a cooperative effect. We also demonstrated that some AP-1 proteins play a different role in terms of Collagenase-3 production according to the stimulator. Indeed, IL-17-induced Collagenase-3 resulted in FosB activation, whereas IL-1β stimulated c-Fos, which may explain the different capacities of these cytokines in producing this enzyme. Our data also suggest that JunB protein plays a rate-limiting step in cytokine-induced Collagenase-3 production in OA chondrocytes. Moreover, we recently identified a novel protein-binding site on the Collagenase-3 promoter that appears to be directly implicated in the repression of its basal transcription. This site was designated AGRE for AG-rich element. This site was not found in other human metalloprotease genes or in the mouse Collagenase. Contrary to the other human Collagenase genes that are transcribed into one mature mRNA, human cells expressed Collagenase-3 transcripts of 3.0, 2.5 and 2.2/2.0 kb as demonstrated by northern blot. We recently identified five different Collagenase-3 RNA species in humans; each could be translated in a cellular environment, indicating that they could be synthesized in response to specific cellular events. For two of the RNA species, the enzyme synthesized would differ from the original Collagenase-3 and will have potentially different function/activity. Moreover, one of the transcripts appears to be an alternative transcription start site. Start sites are known to regulate gene expression by affecting the level of transcription initiation, the translation efficiency of the mRNA produced, and the generation of protein isoforms differing at their amino termini, or may respond differently to the cellular environment. At present, a therapeutic intervention based on the inhibition of metalloproteases is under intensive investigation, and Collagenase-3 appears to be an attractive target for the development of disease-modifying OA drugs. However, the human Collagenase-3 is subjected to different levels of regulation and constitutes a more complex system than originally thought. It would be interesting to verify whether compounds that block only one pathway, such as the synthesis/activity of the original Collagenase-3, are sufficient to block all the effects of this enzyme, or whether all the enzyme transcripts should rather be targeted in order to achieve maximum therapeutic efficacy.

  • Identification and differential expression of human Collagenase-3 mRNA species derived from internal deletion, alternative splicing, and different polyadenylation and transcription initiation sites
    Osteoarthritis and cartilage, 2003
    Co-Authors: Ginette Tardif, Martine Dupuis, Changshan Geng, Pascal Reboul, J.-p. Pelletier, Pierre Ranger, Johanne Martel-pelletier
    Abstract:

    Abstract Objective : Collagenase-3 is a metalloprotease that plays a role in tissue remodeling and pathological processes including arthritis. The human gene is transcribed into major (3.0 and 2.5kb) and minor (2.2/2.0kb) transcripts, as seen in Northern blot assays. We investigated the possibility that other transcripts, not detectable by Northern blot, were synthesized as either coding or regulatory RNAs that would modulate Collagenase-3 expression and function/activity. Design : We screened a cDNA library and total RNA from human chondrocytes by plaque hybridization and RT-PCR, and expressed the transcripts in a cellular environment. The levels of expression of each transcript in normal and osteoarthritic joint cells and cartilage were monitored by RT-PCR. Results : We identified five different Collagenase-3 RNA species derived from alternative polyadenylation sites (COL3-APS), internal deletion (COL3-DEL), alternative splicing (COL3-9B/COL3-9B-2), and different transcription initiation site (COL3-ATS and COL3-ATS-INT). Each transcript could be translated in a cellular environment. Interestingly, the proteins translated from the COL3-DEL and COL3-9B-2 transcripts had a modified hemopexin-like domain, suggesting altered collagenolytic activities. The transcript types COL3-APS, COL3-9B-2, and COL3-ATS were up-regulated in the osteoarthritic samples and expressed in the chondrosarcoma cell line SW1353. Conclusion : Our data show that the human Collagenase-3 gene is subjected to different levels of regulation and constitutes a more complex system than was originally thought.

  • Interleukin 17 (IL-17) induces Collagenase-3 production in human osteoarthritic chondrocytes via AP-1 dependent activation: differential activation of AP-1 members by IL-17 and IL-1beta.
    Journal of Rheumatology, 2002
    Co-Authors: Mohamed Benderdour, Ginette Tardif, Pascal Reboul, Pierre Ranger, Pierre Pelletier, John Di Battista, Johanne Martel-pelletier
    Abstract:

    In osteoarthritic (OA) synovial fluid, many proinflammatory cytokines coexist and stimulate chondrocytes. As interleukin 17 (IL-17) is a catabolic cytokine, we explored its effects on Collagenase-3 production. In a comparative manner we identified IL-17 and IL-1beta induced transcription factors mediating upregulation of this enzyme's production. METHODS: Collagenase-3 levels were determined by ELISA. Transfection experiments of human OA chondrocytes were performed, with the plasmids -1599CAT and -133CAT consisting of 1.6 kb and the first proximal 133 bp containing polyomavirus enhancer A-3 (PEA-3), activating protein-1 (AP-1), and TATA box of the human Collagenase-3 promoter, respectively. Electrophoretic mobility shift assays were done with the AP-1 and PEA-3 oligonucleotides derived from the human Collagenase-3 promoter sequence. Supershift assays were carried out with the specific antibodies against the Jun and Fos proteins. RESULTS: IL-17 induced Collagenase-3 expression and synthesis, with an EC50 at 10 ng/ml. Transfection experiments with wild-type -1599CAT and -133CAT and their mutated AP-1 or PEA-3 derivatives revealed that the AP-1 site was essential for basal and proinflammatory cytokine induced Collagenase-3 promoter activity, whereas the PEA-3 motif exerted a cooperative effect. Of note, in OA chondrocytes, IL-17 and IL-1beta induced Collagenase-3 production through AP-1 occurred with differential protein complexes: IL-17 stimulation resulted in FosB activation, while IL-1beta stimulated c-Fos. Data showed a strong activation of JunB only in cells showing a higher Collagenase-3 basal level and low cytokine (IL-17 and IL-1beta) inducibility, suggesting this transcription factor protein acts as a negative regulator. CONCLUSION: We demonstrated that IL-17 and IL-1beta induced Collagenase-3 production in OA chondrocytes mainly through AP-1 mediated transcriptional activity but with differential protein complexes, suggesting that some AP-1 proteins play a pivotal role in the different cytokine responses in terms of Collagenase-3 production. Our data might suggest that JunB protein plays a rate-limiting step in cytokine induced Collagenase-3 production in OA chondrocytes.

  • A novel negative regulatory element in the human Collagenase-3 proximal promoter region.
    Biochemical and biophysical research communications, 2002
    Co-Authors: Mohamed Benderdour, Ginette Tardif, Jeanpierre Pelletier, Martine Dupuis, Changshan Geng, Johanne Martel-pelletier
    Abstract:

    Abstract We have identified in the human Collagenase-3 promoter a novel negative regulatory element, GAAAAGAAAAAG, designated AGRE (AG-Rich Element). The AGRE site functionality was characterized in human osteoarthritic (OA) chondrocytes as well as four cell lines. The cells were transfected with a plasmid consisting of the first 133 bp of the Collagenase-3 promoter and its AGRE mutated or deleted derivatives. The absence of a functional AGRE site resulted in a statistically significant increase of the Collagenase-3 basal transcription that was not affected by the Collagenase-3 inducers IL-1β and TGF-β1. Two specific protein-AGRE binding complexes were detected by EMSA, and their presence depended on the physiological state of the cell. Indeed, normal chondrocytes and synovial fibroblasts and the four cell lines showed only a slower-migrating complex (complex 1). In OA chondrocytes, the type of complex discriminated two groups—the low-OA chondrocytes, showing low Collagenase-3 basal levels and high inducibility of IL-1β stimulation (complex 1), and the high-OA chondrocytes with high Collagenase-3 basal levels and low IL-1β inducibility (a faster-migrating complex, designated complex 2). UV cross-linking revealed the presence of 48 and 97 kDa proteins in complex 1 and 27, 35, and 73 kDa proteins in complex 2. These findings suggest that the AGRE site plays a rate-limiting role in human Collagenase-3 production.

  • Interleukin 17 (IL-17) induces Collagenase-3 production in human osteoarthritic chondrocytes via AP-1 dependent activation: differential activation of AP-1 members by IL-17 and IL-1beta.
    The Journal of rheumatology, 2002
    Co-Authors: Mohamed Benderdour, Ginette Tardif, Jeanpierre Pelletier, Pascal Reboul, John A. Di Battista, Pierre Ranger, Johanne Martel-pelletier
    Abstract:

    OBJECTIVE: In osteoarthritic (OA) synovial fluid, many proinflammatory cytokines coexist and stimulate chondrocytes. As interleukin 17 (IL-17) is a catabolic cytokine, we explored its effects on Collagenase-3 production. In a comparative manner we identified IL-17 and IL-1beta induced transcription factors mediating upregulation of this enzyme9s production. METHODS: Collagenase-3 levels were determined by ELISA. Transfection experiments of human OA chondrocytes were performed, with the plasmids -1599CAT and -133CAT consisting of 1.6 kb and the first proximal 133 bp containing polyomavirus enhancer A-3 (PEA-3), activating protein-1 (AP-1), and TATA box of the human Collagenase-3 promoter, respectively. Electrophoretic mobility shift assays were done with the AP-1 and PEA-3 oligonucleotides derived from the human Collagenase-3 promoter sequence. Supershift assays were carried out with the specific antibodies against the Jun and Fos proteins. RESULTS: IL-17 induced Collagenase-3 expression and synthesis, with an EC50 at 10 ng/ml. Transfection experiments with wild-type -1599CAT and -133CAT and their mutated AP-1 or PEA-3 derivatives revealed that the AP-1 site was essential for basal and proinflammatory cytokine induced Collagenase-3 promoter activity, whereas the PEA-3 motif exerted a cooperative effect. Of note, in OA chondrocytes, IL-17 and IL-1beta induced Collagenase-3 production through AP-1 occurred with differential protein complexes: IL-17 stimulation resulted in FosB activation, while IL-1beta stimulated c-Fos. Data showed a strong activation of JunB only in cells showing a higher Collagenase-3 basal level and low cytokine (IL-17 and IL-1beta) inducibility, suggesting this transcription factor protein acts as a negative regulator. CONCLUSION: We demonstrated that IL-17 and IL-1beta induced Collagenase-3 production in OA chondrocytes mainly through AP-1 mediated transcriptional activity but with differential protein complexes, suggesting that some AP-1 proteins play a pivotal role in the different cytokine responses in terms of Collagenase-3 production. Our data might suggest that JunB protein plays a rate-limiting step in cytokine induced Collagenase-3 production in OA chondrocytes.

Gillian Murphy - One of the best experts on this subject based on the ideXlab platform.

  • Activation of Progelatinase B (proMMP‐9) by Active Collagenase‐3 (MMP‐13)
    European journal of biochemistry, 1997
    Co-Authors: Vera Knäuper, Carlos López-otín, Bryan D. Smith, Gillian Murphy
    Abstract:

    Human progelatinase B was activated by Collagenase-3 in a time-dependent fashion. Activation proceeded through an intermediate form of Mr 86,000 to the final active form of Mr 82,000. N-terminal amino acid sequence determination demonstrated that the Glu40-Met41 peptide bond was initially hydrolysed followed by cleavage of the Arg87-Phe88 peptide bond releasing the rest of the propeptide domain which was accompanied by the achievement of maximal enzymatic activity as revealed using a quenched fluorescent substrate. Kinetic analysis of activation revealed that the rates were dependent on the concentration of the proenzyme as well as active Collagenase-3. Active gelatinase B did not contribute to the activation rate of the proenzyme initiated by Collagenase-3 and our results indicate that progelatinase B activation proceeds via bimolecular cleavage with Collagenase-3 involving sequential cleavage of the propeptide in two steps. The activation rates were not dependent on C-terminal domain interactions between progelatinase B and Collagenase-3, as assessed using wild-type and C-terminal deletion mutants of both enzymes. Since elevated levels of both gelatinase B and Collagenase-3 have been observed in arthritis and breast cancer pathology these enzymes may well form a proteolytic cascade in these diseases which allows rapid turnover of the extracellular matrix.

  • activation of progelatinase b prommp 9 by active Collagenase 3 mmp 13
    FEBS Journal, 1997
    Co-Authors: Vera Knäuper, Carlos Lopezotin, Bryan D. Smith, Gillian Murphy
    Abstract:

    Human progelatinase B was activated by Collagenase-3 in a time-dependent fashion. Activation proceeded through an intermediate form of Mr 86,000 to the final active form of Mr 82,000. N-terminal amino acid sequence determination demonstrated that the Glu40-Met41 peptide bond was initially hydrolysed followed by cleavage of the Arg87-Phe88 peptide bond releasing the rest of the propeptide domain which was accompanied by the achievement of maximal enzymatic activity as revealed using a quenched fluorescent substrate. Kinetic analysis of activation revealed that the rates were dependent on the concentration of the proenzyme as well as active Collagenase-3. Active gelatinase B did not contribute to the activation rate of the proenzyme initiated by Collagenase-3 and our results indicate that progelatinase B activation proceeds via bimolecular cleavage with Collagenase-3 involving sequential cleavage of the propeptide in two steps. The activation rates were not dependent on C-terminal domain interactions between progelatinase B and Collagenase-3, as assessed using wild-type and C-terminal deletion mutants of both enzymes. Since elevated levels of both gelatinase B and Collagenase-3 have been observed in arthritis and breast cancer pathology these enzymes may well form a proteolytic cascade in these diseases which allows rapid turnover of the extracellular matrix.

  • the role of the c terminal domain of human Collagenase 3 mmp 13 in the activation of proCollagenase 3 substrate specificity and tissue inhibitor of metalloproteinase interaction
    Journal of Biological Chemistry, 1997
    Co-Authors: Vera Knäuper, Bryan J Smith, Susan Cowell, Carlos Lopezotin, Mark Oshea, Helen Morris, Luciano Zardi, Gillian Murphy
    Abstract:

    Abstract Recombinant human proCollagenase-3 and a C-terminal truncated form (Δ249-451 proCollagenase-3) have been stably expressed in myeloma cells and purified. The truncated proenzyme could be processed by aminophenylmercuric acetate via a short-lived intermediate form (N-terminal Leu58) to the final active form (N-terminal Tyr85). The kinetics of activation were not affected by removal of the hemopexin-like C-terminal domain. The specific activities of both Collagenase-3 and Δ249-451 Collagenase-3 were found to be similar using two quenched fluorescent substrates, but Δ249-451 Collagenase-3 failed to cleave native triple helical collagens (types I and II) into characteristic one- and three-quarter fragments. It was noted, however, that the β1,2(I) chains of type I collagen were susceptible to Δ249-451 Collagenase-3, which indicates that the catalytic domain displays telopeptidase activity, thereby generating α1,2(I) chains that are slightly shorter than those in native type I collagen. It can be concluded that the C-terminal domain is only essential for the triple helicase activity of Collagenase-3. Binding of proCollagenase-3 and active Collagenase-3 to type I collagen is mediated by the C-terminal domain. Both Collagenase-3 and Δ249-451 Collagenase-3 hydrolyzed the large tenascin C isoform, fibronectin, recombinant fibronectin fragments, and type IV, IX, X, and XIV collagens; thus, these events were independent from C-terminal domain interactions. In contrast, the minor cartilage type XI collagen was resistant to cleavage. Kinetic analysis of the mechanism of inhibition of wild-type and Δ249-451 Collagenase-3 by wild-type and mutant tissue inhibitors of metalloproteinase (TIMPs) revealed that the association rates for complex formation were influenced by both N- and C-terminal domain interactions. The C-terminal domain of wild-type Collagenase-3 promoted increased association rates with the full-length inhibitors TIMP-1 and TIMP-3 and the hybrid N.TIMP-2/C.TIMP-1 by a factor of up to 33. In contrast, the association rates for complex formation with TIMP-2 and N.TIMP-1/C.TIMP-2 were only marginally affected by C-terminal domain interactions.

  • The role of the C-terminal domain of human Collagenase-3 (MMP-13) in the activation of proCollagenase-3, substrate specificity, and tissue inhibitor of metalloproteinase interaction.
    The Journal of biological chemistry, 1997
    Co-Authors: Vera Knäuper, Susan Cowell, Helen Morris, Luciano Zardi, Carlos López-otín, Bryan D. Smith, Mark O'shea, Gillian Murphy
    Abstract:

    Recombinant human proCollagenase-3 and a C-terminal truncated form (Delta249-451 proCollagenase-3) have been stably expressed in myeloma cells and purified. The truncated proenzyme could be processed by aminophenylmercuric acetate via a short-lived intermediate form (N-terminal Leu58) to the final active form (N-terminal Tyr85). The kinetics of activation were not affected by removal of the hemopexin-like C-terminal domain. The specific activities of both Collagenase-3 and Delta249-451 Collagenase-3 were found to be similar using two quenched fluorescent substrates, but Delta249-451 Collagenase-3 failed to cleave native triple helical collagens (types I and II) into characteristic one- and three-quarter fragments. It was noted, however, that the beta1,2(I) chains of type I collagen were susceptible to Delta249-451 Collagenase-3, which indicates that the catalytic domain displays telopeptidase activity, thereby generating alpha1,2(I) chains that are slightly shorter than those in native type I collagen. It can be concluded that the C-terminal domain is only essential for the triple helicase activity of Collagenase-3. Binding of proCollagenase-3 and active Collagenase-3 to type I collagen is mediated by the C-terminal domain. Both Collagenase-3 and Delta249-451 Collagenase-3 hydrolyzed the large tenascin C isoform, fibronectin, recombinant fibronectin fragments, and type IV, IX, X, and XIV collagens; thus, these events were independent from C-terminal domain interactions. In contrast, the minor cartilage type XI collagen was resistant to cleavage. Kinetic analysis of the mechanism of inhibition of wild-type and Delta249-451 Collagenase-3 by wild-type and mutant tissue inhibitors of metalloproteinase (TIMPs) revealed that the association rates for complex formation were influenced by both N- and C-terminal domain interactions. The C-terminal domain of wild-type Collagenase-3 promoted increased association rates with the full-length inhibitors TIMP-1 and TIMP-3 and the hybrid N.TIMP-2/C.TIMP-1 by a factor of up to 33. In contrast, the association rates for complex formation with TIMP-2 and N.TIMP-1/C.TIMP-2 were only marginally affected by C-terminal domain interactions.

  • The helping hand of Collagenase-3 (MMP-13): 2.7 A crystal structure of its C-terminal haemopexin-like domain.
    Journal of molecular biology, 1996
    Co-Authors: F.x. Gomis-ruth, Gillian Murphy, Vera Knäuper, Carlos López-otín, Ulrich Gohlke, M. Betz, Wolfram Bode
    Abstract:

    Abstract Collagenase-3 (MMP-13) is a matrix metalloproteinase involved in human breast cancer pathology and in arthritic processes. The crystal structure of its C-terminal haemopexin-like domain has been solved by molecular replacement and refined to an R -value of 0.195 using data to 2.7 A resolution. This structure reveals a disk-like shape. The chain is folded into a β-propeller structure of pseudo 4-fold symmetry, with the four propeller blades arranged around a funnel-like tunnel. This central tunnel tube harbours four ions assigned as two calcium and two chloride ions. The C-terminal domain of Collagenase-3 has a similar structure to the equivalent domain of gelatinase A and fibroblast Collagenase 1; however, its detailed structure and surface charge pattern has a somewhat greater similarity to the latter, in agreement with the subgrouping of MMP-13 with the Collagenase subfamily of MMPs. It is proposed that several small structural differences may act together to confer the characteristic binding and cleavage specificities of Collagenases for triple-helical substrates, probably in co-operation with a fitting interdomain linker.

Vera Knäuper - One of the best experts on this subject based on the ideXlab platform.

  • Activation of Progelatinase B (proMMP‐9) by Active Collagenase‐3 (MMP‐13)
    European journal of biochemistry, 1997
    Co-Authors: Vera Knäuper, Carlos López-otín, Bryan D. Smith, Gillian Murphy
    Abstract:

    Human progelatinase B was activated by Collagenase-3 in a time-dependent fashion. Activation proceeded through an intermediate form of Mr 86,000 to the final active form of Mr 82,000. N-terminal amino acid sequence determination demonstrated that the Glu40-Met41 peptide bond was initially hydrolysed followed by cleavage of the Arg87-Phe88 peptide bond releasing the rest of the propeptide domain which was accompanied by the achievement of maximal enzymatic activity as revealed using a quenched fluorescent substrate. Kinetic analysis of activation revealed that the rates were dependent on the concentration of the proenzyme as well as active Collagenase-3. Active gelatinase B did not contribute to the activation rate of the proenzyme initiated by Collagenase-3 and our results indicate that progelatinase B activation proceeds via bimolecular cleavage with Collagenase-3 involving sequential cleavage of the propeptide in two steps. The activation rates were not dependent on C-terminal domain interactions between progelatinase B and Collagenase-3, as assessed using wild-type and C-terminal deletion mutants of both enzymes. Since elevated levels of both gelatinase B and Collagenase-3 have been observed in arthritis and breast cancer pathology these enzymes may well form a proteolytic cascade in these diseases which allows rapid turnover of the extracellular matrix.

  • activation of progelatinase b prommp 9 by active Collagenase 3 mmp 13
    FEBS Journal, 1997
    Co-Authors: Vera Knäuper, Carlos Lopezotin, Bryan D. Smith, Gillian Murphy
    Abstract:

    Human progelatinase B was activated by Collagenase-3 in a time-dependent fashion. Activation proceeded through an intermediate form of Mr 86,000 to the final active form of Mr 82,000. N-terminal amino acid sequence determination demonstrated that the Glu40-Met41 peptide bond was initially hydrolysed followed by cleavage of the Arg87-Phe88 peptide bond releasing the rest of the propeptide domain which was accompanied by the achievement of maximal enzymatic activity as revealed using a quenched fluorescent substrate. Kinetic analysis of activation revealed that the rates were dependent on the concentration of the proenzyme as well as active Collagenase-3. Active gelatinase B did not contribute to the activation rate of the proenzyme initiated by Collagenase-3 and our results indicate that progelatinase B activation proceeds via bimolecular cleavage with Collagenase-3 involving sequential cleavage of the propeptide in two steps. The activation rates were not dependent on C-terminal domain interactions between progelatinase B and Collagenase-3, as assessed using wild-type and C-terminal deletion mutants of both enzymes. Since elevated levels of both gelatinase B and Collagenase-3 have been observed in arthritis and breast cancer pathology these enzymes may well form a proteolytic cascade in these diseases which allows rapid turnover of the extracellular matrix.

  • the role of the c terminal domain of human Collagenase 3 mmp 13 in the activation of proCollagenase 3 substrate specificity and tissue inhibitor of metalloproteinase interaction
    Journal of Biological Chemistry, 1997
    Co-Authors: Vera Knäuper, Bryan J Smith, Susan Cowell, Carlos Lopezotin, Mark Oshea, Helen Morris, Luciano Zardi, Gillian Murphy
    Abstract:

    Abstract Recombinant human proCollagenase-3 and a C-terminal truncated form (Δ249-451 proCollagenase-3) have been stably expressed in myeloma cells and purified. The truncated proenzyme could be processed by aminophenylmercuric acetate via a short-lived intermediate form (N-terminal Leu58) to the final active form (N-terminal Tyr85). The kinetics of activation were not affected by removal of the hemopexin-like C-terminal domain. The specific activities of both Collagenase-3 and Δ249-451 Collagenase-3 were found to be similar using two quenched fluorescent substrates, but Δ249-451 Collagenase-3 failed to cleave native triple helical collagens (types I and II) into characteristic one- and three-quarter fragments. It was noted, however, that the β1,2(I) chains of type I collagen were susceptible to Δ249-451 Collagenase-3, which indicates that the catalytic domain displays telopeptidase activity, thereby generating α1,2(I) chains that are slightly shorter than those in native type I collagen. It can be concluded that the C-terminal domain is only essential for the triple helicase activity of Collagenase-3. Binding of proCollagenase-3 and active Collagenase-3 to type I collagen is mediated by the C-terminal domain. Both Collagenase-3 and Δ249-451 Collagenase-3 hydrolyzed the large tenascin C isoform, fibronectin, recombinant fibronectin fragments, and type IV, IX, X, and XIV collagens; thus, these events were independent from C-terminal domain interactions. In contrast, the minor cartilage type XI collagen was resistant to cleavage. Kinetic analysis of the mechanism of inhibition of wild-type and Δ249-451 Collagenase-3 by wild-type and mutant tissue inhibitors of metalloproteinase (TIMPs) revealed that the association rates for complex formation were influenced by both N- and C-terminal domain interactions. The C-terminal domain of wild-type Collagenase-3 promoted increased association rates with the full-length inhibitors TIMP-1 and TIMP-3 and the hybrid N.TIMP-2/C.TIMP-1 by a factor of up to 33. In contrast, the association rates for complex formation with TIMP-2 and N.TIMP-1/C.TIMP-2 were only marginally affected by C-terminal domain interactions.

  • The role of the C-terminal domain of human Collagenase-3 (MMP-13) in the activation of proCollagenase-3, substrate specificity, and tissue inhibitor of metalloproteinase interaction.
    The Journal of biological chemistry, 1997
    Co-Authors: Vera Knäuper, Susan Cowell, Helen Morris, Luciano Zardi, Carlos López-otín, Bryan D. Smith, Mark O'shea, Gillian Murphy
    Abstract:

    Recombinant human proCollagenase-3 and a C-terminal truncated form (Delta249-451 proCollagenase-3) have been stably expressed in myeloma cells and purified. The truncated proenzyme could be processed by aminophenylmercuric acetate via a short-lived intermediate form (N-terminal Leu58) to the final active form (N-terminal Tyr85). The kinetics of activation were not affected by removal of the hemopexin-like C-terminal domain. The specific activities of both Collagenase-3 and Delta249-451 Collagenase-3 were found to be similar using two quenched fluorescent substrates, but Delta249-451 Collagenase-3 failed to cleave native triple helical collagens (types I and II) into characteristic one- and three-quarter fragments. It was noted, however, that the beta1,2(I) chains of type I collagen were susceptible to Delta249-451 Collagenase-3, which indicates that the catalytic domain displays telopeptidase activity, thereby generating alpha1,2(I) chains that are slightly shorter than those in native type I collagen. It can be concluded that the C-terminal domain is only essential for the triple helicase activity of Collagenase-3. Binding of proCollagenase-3 and active Collagenase-3 to type I collagen is mediated by the C-terminal domain. Both Collagenase-3 and Delta249-451 Collagenase-3 hydrolyzed the large tenascin C isoform, fibronectin, recombinant fibronectin fragments, and type IV, IX, X, and XIV collagens; thus, these events were independent from C-terminal domain interactions. In contrast, the minor cartilage type XI collagen was resistant to cleavage. Kinetic analysis of the mechanism of inhibition of wild-type and Delta249-451 Collagenase-3 by wild-type and mutant tissue inhibitors of metalloproteinase (TIMPs) revealed that the association rates for complex formation were influenced by both N- and C-terminal domain interactions. The C-terminal domain of wild-type Collagenase-3 promoted increased association rates with the full-length inhibitors TIMP-1 and TIMP-3 and the hybrid N.TIMP-2/C.TIMP-1 by a factor of up to 33. In contrast, the association rates for complex formation with TIMP-2 and N.TIMP-1/C.TIMP-2 were only marginally affected by C-terminal domain interactions.

  • The helping hand of Collagenase-3 (MMP-13): 2.7 A crystal structure of its C-terminal haemopexin-like domain.
    Journal of molecular biology, 1996
    Co-Authors: F.x. Gomis-ruth, Gillian Murphy, Vera Knäuper, Carlos López-otín, Ulrich Gohlke, M. Betz, Wolfram Bode
    Abstract:

    Abstract Collagenase-3 (MMP-13) is a matrix metalloproteinase involved in human breast cancer pathology and in arthritic processes. The crystal structure of its C-terminal haemopexin-like domain has been solved by molecular replacement and refined to an R -value of 0.195 using data to 2.7 A resolution. This structure reveals a disk-like shape. The chain is folded into a β-propeller structure of pseudo 4-fold symmetry, with the four propeller blades arranged around a funnel-like tunnel. This central tunnel tube harbours four ions assigned as two calcium and two chloride ions. The C-terminal domain of Collagenase-3 has a similar structure to the equivalent domain of gelatinase A and fibroblast Collagenase 1; however, its detailed structure and surface charge pattern has a somewhat greater similarity to the latter, in agreement with the subgrouping of MMP-13 with the Collagenase subfamily of MMPs. It is proposed that several small structural differences may act together to confer the characteristic binding and cleavage specificities of Collagenases for triple-helical substrates, probably in co-operation with a fitting interdomain linker.