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The Journal of Biological Chemistry Jun 2003ADAMs are membrane-anchored glycoproteins with functions in fertilization, heart development, neurogenesis, and protein ectodomain shedding. Here we report an evaluation...
ADAMs are membrane-anchored glycoproteins with functions in fertilization, heart development, neurogenesis, and protein ectodomain shedding. Here we report an evaluation of the catalytic activity of recombinantly expressed soluble forms of ADAM19, a protein that is essential for cardiovascular morphogenesis. Proteolytic activity of soluble forms of ADAM19 was first demonstrated by their autocatalytic removal of a purification tag (Myc-His) and their ability to cleave myelin basic protein and the insulin B chain. The metalloprotease activity of ADAM19 is sensitive to the hydroxamic acid-type metalloprotease inhibitor BB94 (batimastat) but not to tissue inhibitors of metalloproteases (TIMPs) 1-3. Moreover, ADAM19 cleaves peptides corresponding to the known cleavage sites of tumor necrosis factor-alpha (TNF-alpha), TNF-related activation-induced cytokine (TRANCE, also referred to as osteoprotegerin ligand), and kit ligand-1 (KL-1) in vitro. Although ADAM19 is not required for shedding of TNFalpha and TRANCE in mouse embryonic fibroblasts, its overexpression in COS-7 cells results in strongly increased TRANCE shedding. This suggests a potential role for ADAM19 in shedding TRANCE in cells where both molecules are highly expressed, such as in osteoblasts. Interestingly, our results also indicate that ADAM19 can function as a negative regulator of KL-1 shedding in both COS-7 cells and mouse embryonic fibroblasts, instead of acting directly on KL-1. The identification of potential in vitro substrates offers the basis for further functional studies of ADAM19 in cells and in mice.
Topics: ADAM Proteins; Amino Acid Sequence; Animals; Binding Sites; COS Cells; Catalysis; Cell Line; Chlorocebus aethiops; Disintegrins; Fibroblasts; Kinetics; Membrane Proteins; Metalloendopeptidases; Metalloproteases; Mice; Mice, Knockout; Molecular Sequence Data; Muscle Proteins; Recombinant Proteins; Spodoptera; Transfection
PubMed: 12682046
DOI: 10.1074/jbc.M302781200 -
Mediators of Inflammation 2014The chemokine fractalkine is considered as unique since it exists both as membrane-bound adhesion molecule and as shed soluble chemoattractant. Here the hypothesis was...
The chemokine fractalkine is considered as unique since it exists both as membrane-bound adhesion molecule and as shed soluble chemoattractant. Here the hypothesis was tested whether placental fractalkine can be shed and released into the maternal circulation. Immunohistochemical staining of human first trimester and term placenta sections localized fractalkine at the apical microvillous plasma membrane of the syncytiotrophoblast. Gene expression analysis revealed abundant upregulation in placental fractalkine at term, compared to first trimester. Fractalkine expression and release were detected in the trophoblast cell line BeWo, in primary term trophoblasts and placental explants. Incubation of BeWo cells and placental explants with metalloprotease inhibitor Batimastat inhibited the release of soluble fractalkine and at the same time increased the membrane-bound form. These results demonstrate that human placenta is a source for fractalkine, which is expressed in the syncytiotrophoblast and can be released into the maternal circulation by constitutive metalloprotease dependent shedding. Increased expression and release of placental fractalkine may contribute to low grade systemic inflammatory responses in third trimester of normal pregnancy. Aberrant placental metalloprotease activity may not only affect the release of placenta derived fractalkine but may at the same time affect the abundance of the membrane-bound form of the chemokine.
Topics: Adult; Cell Line; Cell Membrane; Chemokine CX3CL1; Female; Gene Expression Regulation, Enzymologic; Humans; Immunohistochemistry; Inflammation; Metalloproteases; Microvilli; Phenylalanine; Placenta; Pregnancy; Pregnancy Trimester, First; Thiophenes; Trophoblasts
PubMed: 24771984
DOI: 10.1155/2014/839290 -
Acta Crystallographica. Section F,... Jun 2012The role of ADAM-8 in cancer and inflammatory diseases such as allergy, arthritis and asthma makes it an attractive target for drug development. Therefore, the catalytic...
The role of ADAM-8 in cancer and inflammatory diseases such as allergy, arthritis and asthma makes it an attractive target for drug development. Therefore, the catalytic domain of human ADAM-8 was expressed, purified and crystallized in complex with a hydroxamic acid inhibitor, batimastat. The crystal structure of the enzyme-inhibitor complex was refined to 2.1 Å resolution. ADAM-8 has an overall fold similar to those of other ADAM members, including a central five-stranded β-sheet and a catalytic Zn(2+) ion. However, unique differences within the S1' binding loop of ADAM-8 are observed which might be exploited to confer specificity and selectivity to ADAM-8 competitive inhibitors for the treatment of diseases involving this enzyme.
Topics: ADAM Proteins; Catalytic Domain; Humans; Ligands; Membrane Proteins; Models, Molecular; Phenylalanine; Protease Inhibitors; Protein Binding; Protein Unfolding; Thiophenes
PubMed: 22684055
DOI: 10.1107/S1744309112015618 -
Annals of Oncology : Official Journal... Dec 1995Matrix metalloproteinases are a homologous family of proteolytic enzymes. Collectively, these proteinases are capable of degrading all components of the extracellular... (Review)
Review
BACKGROUND
Matrix metalloproteinases are a homologous family of proteolytic enzymes. Collectively, these proteinases are capable of degrading all components of the extracellular matrix, including proteolytically resistant fibrillar collagens. Extracellular matrices constitute the principal barrier to tumour growth and spread, and there is now experimental evidence that malignant tumours utilise matrix metalloproteinases to overcome this barrier. Inhibitors of matrix metalloproteinases may therefore be of therapeutic value in the treatment of metastatic disease.
DESIGN
This review describes the activity of matrix metalloproteinases inhibitors (MMPIs), in experimental tumour models and in phase I/II clinical studies.
RESULTS
Studies with MMPIs in vitro have shown that these agents are not cytotoxic but can inhibit the degradation of extracellular matrix by tumour cells. In experimental tumour models in vivo, MMPI treatment caused inhibition of tumour growth and metastatic spread in both rodent syngeneic and human xenograft models. MMPIs have also been shown to inhibit angiogenesis, a process essential for the rapid growth of most malignancies.
CONCLUSIONS
MMPI therapy has the potential to arrest tumour growth and spread. As a non-cytotoxic 'tumourostatic' approach it may offer an ideal complement to surgery, radiotherapy and chemotherapy in the successful long-term treatment of metastatic disease.
Topics: Animals; Antineoplastic Agents; Clinical Trials as Topic; Humans; Melanoma, Experimental; Metalloendopeptidases; Neovascularization, Pathologic; Phenylalanine; Protease Inhibitors; Thiophenes
PubMed: 8750146
DOI: 10.1093/oxfordjournals.annonc.a059091 -
Scientific Reports Jul 2017In atherosclerosis, matrix metallopeptidases (MMPs) contribute to plaque rupture through weakening of the fibrous cap. Pleiotropic P2X purinoceptor 7 (P2X7), expressed...
In atherosclerosis, matrix metallopeptidases (MMPs) contribute to plaque rupture through weakening of the fibrous cap. Pleiotropic P2X purinoceptor 7 (P2X7), expressed in the carotid plaque (PL), is involved in interleukin 1 beta (IL-1β) release that may influence MMP9 generation, thus their possible modulation through acting on P2X7 was investigated. P2X7-related machinery was characterized and the effects of P2X7 antagonists (A740003, KN62) and MMPs inhibitors (Batimastat, Ro28-2653) were studied in ex-vivo tissue cultures of human PL's vs. non-atherosclerotic internal mammary artery (IMA) by using molecular biology, immune-biochemical and microscopy methodologies. We highlighted atherosclerosis-related differences between PLs and IMAs molecular patterns, and their responsivity to P2X7 antagonism. High IL-1β tissue content was associated with PLs morphology and instability/vulnerability. We demonstrated that A740003, but not KN62, decreased IL-1β and MMP9 independently from NLR family pyrin domain containing 3, but in relationship with patient's smoking status. Acting downstream P2X7 by MMPs inhibitors, diminished IL-1β mRNA without transcriptional effect at MMP9, possibly because the assumption of statin by patients. These data firstly demonstrated A740003 suitability as a specific tool to decrease inflammatory status in human vessels and might support the design of studies applying P2X7 antagonists for the local targeting and tailored therapy of atherosclerosis.
Topics: Atherosclerosis; Female; Humans; Immunohistochemistry; Inflammation; Interleukin-1beta; Male; Matrix Metalloproteinase 9; Microscopy, Fluorescence; Organ Culture Techniques; Pathology, Molecular; Receptors, Purinergic P2X7
PubMed: 28687781
DOI: 10.1038/s41598-017-05137-y -
EMBO Molecular Medicine Oct 2017We have characterised the proteolytic cleavage events responsible for the shedding of triggering receptor expressed on myeloid cells 2 (TREM2) from primary cultures of...
We have characterised the proteolytic cleavage events responsible for the shedding of triggering receptor expressed on myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2-expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N-terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site, and mass spectrometry confirmed that shedding occurred predominantly at the H157-S158 peptide bond for both wild-type and H157Y human TREM2 and for the wild-type murine orthologue. Crucially, we also show that the Alzheimer's disease-associated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat- and ADAM10-siRNA-independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer's and other neurological diseases.
Topics: ADAM10 Protein; ADAM17 Protein; Alzheimer Disease; Amyloid Precursor Protein Secretases; Animals; Animals, Newborn; Culture Media, Conditioned; HEK293 Cells; Humans; Ketocholesterols; Macrophages; Matrix Metalloproteinase Inhibitors; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Microglia; Primary Cell Culture; Proteolysis; RNA, Small Interfering; Receptors, Immunologic
PubMed: 28855301
DOI: 10.15252/emmm.201707673 -
International Journal of Hyperthermia :... 2002We investigated the anti-vascular activity of local hyperthermia (44 degrees C, 60 min) in s.c. BT4An rat gliomas, and the influence on tumour growth of hyperthermia and...
We investigated the anti-vascular activity of local hyperthermia (44 degrees C, 60 min) in s.c. BT4An rat gliomas, and the influence on tumour growth of hyperthermia and the anti-angiogenic compound batimastat (30 mg/kg i.p.). Heat-induced vascular damage was assessed in small (82 mm3) and large (171 mm3) tumours using confocal microscopy and immunostaining for von Willebrand factor. The influence of hyperthermia on tumour blood flow was measured using the 86RbCl method. The anti-tumour activity of hyperthermia (one or two fractions a week apart) and batimastat (7 or 14 daily injections) and various combination schedules were examined. Hyperthermia disrupted 25-50% of the vasculature in the BT4An tumours, and the vascular damage was most extensive in the central part of the large tumours. The heated tumours exhibited a 40-60% blood flow reduction, which persisted until the last measurement after 24 h. One fraction of hyperthermia caused a significant growth delay of 4 days, compared with the control group (p = 0.01), but no additional tumour response was produced by a second heating session. Batimastat had no influence on tumour growth and the combination of drug and local heating did not enhance the tumour response, compared with heating alone. It was concluded that hyperthermia at 44 degrees C for 60min exhibits anti-vascular activity and inhibits tumour growth in the BT4An tumour model. Batimastat had no effect on tumour growth.
Topics: Angiogenesis Inhibitors; Animals; Brain Neoplasms; Female; Glioma; Hyperthermia, Induced; Male; Phenylalanine; Rats; Thiophenes
PubMed: 11911484
DOI: 10.1080/02656730110090712 -
Annales de Biologie Clinique 2003Matrix metalloproteinases (MMPs) play a key role in the physiology of connective tissue development, morphogenesis and wound healing, but their unregulated activity has... (Comparative Study)
Comparative Study Review
Matrix metalloproteinases (MMPs) play a key role in the physiology of connective tissue development, morphogenesis and wound healing, but their unregulated activity has been implicated in numerous disease processes including arthritis, tumor cell metastasis and atherosclerosis. MMP family consists of at least 20 members; MMPs are produced by the different cell types (vascular smooth muscle cells, monocytes, endothelial cells) involved in the atheromatous plaque formation and participate to extracellular matrix remodelling and cell infiltration or migration. Since excessive tissue remodelling and increased matrix metalloproteinase activity have been demonstrated during atherosclerotic lesion progression (including plaque disruption), MMPs represent a potential target for therapeutic intervention to modify vascular pathology, by restoring the MMP/TIMP physiological equilibrium. This review highlights the structures of MMPs and their physiological inhibitors, the Tissue Inhibitors of MMPs (TIMPs), and describes the current developments in pharmacological MMP inhibition.
Topics: Animals; Anti-Bacterial Agents; Antineoplastic Agents; Aortic Diseases; Arteriosclerosis; Case-Control Studies; Clinical Trials as Topic; Coronary Artery Disease; Doxycycline; Humans; Hydroxamic Acids; Hyperlipidemias; Hypolipidemic Agents; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Metalloendopeptidases; Organic Chemicals; Phenylalanine; Polymorphism, Genetic; Prospective Studies; Rats; Risk Factors; Thiophenes; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinase-3; Tissue Inhibitor of Metalloproteinases
PubMed: 12702469
DOI: No ID Found -
International Journal of Molecular... Sep 2020The crucial role of extracellular proteases in cancer progression is well-known, especially in relation to the promotion of cell invasion through extracellular matrix... (Review)
Review
The crucial role of extracellular proteases in cancer progression is well-known, especially in relation to the promotion of cell invasion through extracellular matrix remodeling. This also occurs by the ability of extracellular proteases to induce the shedding of transmembrane proteins at the plasma membrane surface or within extracellular vesicles. This process results in the regulation of key signaling pathways by the modulation of kinases, e.g., the epidermal growth factor receptor (EGFR). Considering their regulatory roles in cancer, therapeutics targeting various extracellular proteases have been discovered. These include the metal-binding agents di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which increase c-MET degradation by multiple mechanisms. Both the direct and indirect inhibition of protease expression and activity can be achieved through metal ion depletion. Considering direct mechanisms, chelators can bind zinc(II) that plays a catalytic role in enzyme activity. In terms of indirect mechanisms, Dp44mT and DpC potently suppress the expression of the kallikrein-related peptidase-a prostate-specific antigen-in prostate cancer cells. The mechanism of this activity involves promotion of the degradation of the androgen receptor. Additional suppressive mechanisms of Dp44mT and DpC on matrix metalloproteases (MMPs) relate to their ability to up-regulate the metastasis suppressors N-myc downstream regulated gene-1 (NDRG1) and NDRG2, which down-regulate MMPs that are crucial for cancer cell invasion.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Transformation, Neoplastic; Chelating Agents; Disease Progression; Drug Design; Drug Screening Assays, Antitumor; Extracellular Fluid; Extracellular Vesicles; Humans; Hydroxamic Acids; Iron; Iron Chelating Agents; Kallikreins; Matrix Metalloproteinases; Molecular Targeted Therapy; Neoplasm Proteins; Oxaprozin; Peptide Hydrolases; Phenylalanine; Protease Inhibitors; Protein Kinases; Pyridines; Thiophenes; Thiosemicarbazones; Zinc
PubMed: 32948029
DOI: 10.3390/ijms21186805 -
Experimental Eye Research Apr 2014Previous studies have shown that platelet derived growth factor (PDGF) can stimulate corneal keratocyte spreading and migration within 3-D collagen matrices, without...
Previous studies have shown that platelet derived growth factor (PDGF) can stimulate corneal keratocyte spreading and migration within 3-D collagen matrices, without inducing transformation to a contractile, fibroblastic phenotype. The goal of this study was to investigate the role of matrix metalloproteinases (MMPs) in regulating PDGF-induced changes in keratocyte motility and mechanical differentiation. Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-) to maintain their quiescent phenotype. A nested collagen matrix construct was used to assess 3-D cell migration, and a standard collagen matrix model was used to assess cell morphology and cell-mediated matrix contraction. In both cases constructs were cultured in S- supplemented with PDGF, with or without the broad spectrum MMP inhibitors GM6001 or BB-94. After 4 days, f-actin, nuclei and collagen fibrils were imaged using confocal microscopy. To assess sub-cellular mechanical activity (extension and retraction of cell processes), time-lapse DIC imaging was also performed. MT1-MMP expression and MMP-mediated collagen degradation were also examined. Results demonstrated that neither GM6001 nor BB-94 affected corneal keratocyte viability or proliferation in 3-D culture. PDGF stimulated elongation and migration of corneal keratocytes within type I collagen matrices, without causing a loss of their dendritic morphology or inducing formation of intracellular stress fibers. Treatment with GM6001 and BB-94 inhibited PDGF-induced keratocyte spreading and migration. Relatively low levels of keratocyte-induced matrix contraction were also maintained in PDGF, and the amount of PDGF-induced collagen degradation was similar to that observed in S- controls. The collagen degradation pattern was consistent with membrane-associated MMP activity, and keratocytes showed positive staining for MT1-MMP, albeit weak. Both matrix contraction and collagen degradation were reduced by MMP inhibition. For most outcome measures, the inhibitory effect of BB-94 was significantly greater than that of GM6001. Overall, the data demonstrate for the first time that even under conditions in which low levels of contractility and extracellular matrix proteolysis are maintained, MMPs still play an important role in mediating cell spreading and migration within 3-D collagen matrices. This appears to be mediated at least in part by membrane-tethered MMPs, such as MT1-MMP.
Topics: Actins; Animals; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Collagen Type I; Corneal Keratocytes; Culture Media, Serum-Free; Dipeptides; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Microscopy, Confocal; Phenylalanine; Platelet-Derived Growth Factor; Rabbits; Thiophenes
PubMed: 24530619
DOI: 10.1016/j.exer.2014.02.002