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Philip C Trackman - One of the best experts on this subject based on the ideXlab platform.
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A Novel Function for Lysyl Oxidase in Pluripotent Mesenchymal Cell Proliferation and Relevance to Inflammation-Associated Osteopenia
PloS one, 2014Co-Authors: Roozbeh Khosravi, Katharine L. Sodek, Manish V. Bais, Debashree Saxena, Michael Faibish, Philip C TrackmanAbstract:Lysyl Oxidase is a multifunctional enzyme required for collagen biosynthesis. Various growth factors regulate Lysyl Oxidase during osteoblast differentiation, subject to modulation by cytokines such as TNF-α in inflammatory osteopenic disorders including diabetic bone disease. Canonical Wnt signaling promotes osteoblast development. Here we investigated the effect of Wnt3a and TNF-α on Lysyl Oxidase expression in pluripotent C3H10T1/2 cells, bone marrow stromal cells, and committed osteoblasts. Lysyl Oxidase was up-regulated by a transcriptional mechanism 3-fold in C3H10T1/2 cells, and 2.5-fold in bone marrow stromal cells. A putative functional TCF/LEF element was identified in the Lysyl Oxidase promoter. Interestingly, Lysyl Oxidase was not up-regulated in committed primary rat calvarial- or MC3T3-E1 osteoblasts. TNF-α down-regulated Lysyl Oxidase both in Wnt3a-treated and in non-treated C3H10T1/2 cells by a post-transcriptional mechanism mediated by miR203. Non-differentiated cells do not produce a collagen matrix; thus, a novel biological role for Lysyl Oxidase in pluripotent cells was investigated. Lysyl Oxidase shRNAs effectively silenced Lysyl Oxidase expression, and suppressed the growth of C3H10T1/2 cells by 50%, and blocked osteoblast differentiation. We propose that interference with Lysyl Oxidase expression under excess inflammatory conditions such as those that occur in diabetes, osteoporosis, or rheumatoid arthritis can result in a diminished pool of pluripotent cells which ultimately contributes to osteopenia.
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collagen advanced glycation inhibits its discoidin domain receptor 2 ddr2 mediated induction of Lysyl Oxidase in osteoblasts
Bone, 2014Co-Authors: Roozbeh Khosravi, Katharine L. Sodek, Michael Faibish, Philip C TrackmanAbstract:Abstract Diabetes increases the risk of bone fracture. Organic and inorganic bone extracellular matrix components determine bone strength. Previous studies indicate that in diabetes, glycation of collagen causes abnormal arrangements of collagen molecules and fragile bones. Diabetic bone fragility is additionally attributed to reduced levels of Lysyl Oxidase enzyme-dependent collagen cross-links. The mechanism underlying the presence of lower enzymatic collagen cross-links in diabetic bone has not been directly investigated. Here we determine in primary osteoblast cultures the regulation of Lysyl Oxidase protein by type I collagen and collagen modified by carboxymethylation (CML-collagen), a form of advanced glycation endproducts. Data indicate that non-glycated collagen up-regulates Lysyl Oxidase levels both in primary non-differentiated and in differentiating mouse and rat osteoblast cultures, while CML-collagen fails to regulate Lysyl Oxidase in these cells. Collagen binding to Discoidin Domain Receptor-2 (DDR2) mediates Lysyl Oxidase increases, determined in DDR2 shRNA knockdown studies. DDR2 binding and activation were disrupted by collagen glycation, pointing to a mechanism for the diminished levels of Lysyl Oxidase and consequently low Lysyl Oxidase-derived cross-links in diabetic bone. Our studies indicate that collagen–integrin interactions may not play a major role in up-regulating Lysyl Oxidase. Furthermore, non-collagenous ligands for the receptor for advanced glycation end products (RAGE) failed to alter Lysyl Oxidase levels. Taken together with published studies a new understanding emerges in which diabetes- and age-dependent inhibition of normal collagen-stimulated DDR2- and integrin-signaling, and independent advanced glycation-stimulated RAGE-signaling, each contributes to different aspects of diabetic osteopenia.
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Characterization of Recombinant Lysyl Oxidase Propeptide
Biochemistry, 2010Co-Authors: Siddharth R. Vora, Ying Guo, Danielle Stephens, Erdjan Salih, Kathrin H. Kirsch, Gail E. Sonenshein, Philip C TrackmanAbstract:Lysyl Oxidase enzyme activity is critical for the biosynthesis of mature and functional collagens and elastin. In addition, Lysyl Oxidase has tumor suppressor activity that has been shown to depend on the propeptide region (LOX-PP) derived from pro-Lysyl Oxidase (Pro-LOX), and not on Lysyl Oxidase enzyme activity. Pro-LOX is secreted as a 50 kDa proenzyme, and then undergoes biosynthetic proteolytic processing to active ~30 kDa LOX enzyme and LOX-PP. The present study reports the efficient recombinant expression and purification of rat LOX-PP. Moreover, using enzymatic deglycosylation and DTT derivatization combined with mass spectrometry technologies, it is shown for the first time that rLOX-PP and naturally occurring LOX-PP contain both N- and O-linked carbohydrates. Structure predictions furthermore suggest that LOX-PP is a mostly disordered protein, which was experimentally confirmed in circular dichroism studies. Due to its high isoelectric point and its disordered structure, we propose that LOX-PP can associate with extracellular and intracellular binding partners to affect its known biological activities as a tumor suppressor and inhibitor of cell proliferation.
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The Propeptide Domain of Lysyl Oxidase Induces Phenotypic Reversion of Ras-transformed Cells
The Journal of biological chemistry, 2004Co-Authors: Amitha H Palamakumbura, Ying Guo, Gail E. Sonenshein, P. Sommer, S. Jeay, N. Pischon, Philip C TrackmanAbstract:Abstract Lysyl Oxidase is an extracellular enzyme critical for the normal biosynthesis of collagens and elastin. In addition, Lysyl Oxidase reverts ras-mediated transformation, and Lysyl Oxidase expression is down-regulated in human cancers. Since suramin inhibits growth factor signaling pathways and induces Lysyl Oxidase in ras-transformed NIH3T3 cells (RS485 cells), we sought to investigate the effects of suramin on the phenotype of transformed cells and the role of Lysyl Oxidase in mediating these effects. Suramin treatment resulted in a more normal phenotype as judged by growth rate, cell cycle parameters, and morphology. β-aminopropionitrile, the selective inhibitor of Lysyl Oxidase enzyme activity, was remarkably unable to block suramin-induced reversion. By contrast, ectopic antisense Lysyl Oxidase demonstrated that Lysyl Oxidase gene expression mediated phenotypic reversion. Since Lysyl Oxidase is synthesized as a 50 kDa precursor and processed to a 30 kDa active enzyme and 18 kDa propeptide, the effects of these two products on the transformed phenotype of RS485 cells were then directly assessed in the absence of suramin. Here we report, for the first time, that the Lysyl Oxidase propeptide, and not the Lysyl Oxidase enzyme, inhibits ras-dependent transformation as determined by effects on cell proliferation assays, growth in soft agar, and Akt-dependent induction of NF-κB activity. Thus, the Lysyl Oxidase propeptide, which is released during extracellular proteolytic processing of pro-Lysyl Oxidase, functions to inhibit ras-dependent cell transformation.
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Autocrine growth factor regulation of Lysyl Oxidase expression in transformed fibroblasts.
The Journal of biological chemistry, 2003Co-Authors: Amitha H Palamakumbura, P. Sommer, Philip C TrackmanAbstract:Lysyl Oxidase catalyzes oxidative deamination of peptidyl-lysine and hydroxylysine residues in collagens and lysine residues in elastin to form peptidyl aldehydes that are required for the formation of covalent cross-links in normal extracellular matrix biosynthesis. Lysyl Oxidase in addition has tumor suppressor activity, and phenotypic reversion of transformed cell lines is accompanied by increased Lysyl Oxidase expression. The mechanism of low expression of Lysyl Oxidase in tumor cells is unknown. The present study investigates the hypothesis that autocrine growth factor pathways maintain low Lysyl Oxidase expression levels in c-H-ras-transformed fibroblasts (RS485 cell line). Autocrine pathways were blocked with suramin, a general inhibitor of growth factor receptor binding, and resulted in more than a 10-fold increase in Lysyl Oxidase expression and proenzyme production. This regulation was found to be reversible and occurred at the transcriptional level determined using Lysyl Oxidase promoter/reporter gene assays. Function blocking anti-fibroblast growth factor-2 (FGF-2) antibody enhanced Lysyl Oxidase expression in the absence of suramin. Finally, the addition of FGF-2 to suramin-treated cells completely reversed suramin stimulation of Lysyl Oxidase mRNA levels. Data support that an FGF-2 autocrine pathway inhibits Lysyl Oxidase transcription in the tumorigenic-transformed RS485 cell line. This finding may be of therapeutic significance and, in addition, provides a new experimental approach to investigate the mechanism of the tumor suppressor activity of Lysyl Oxidase.
Herbert M. Kagan - One of the best experts on this subject based on the ideXlab platform.
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Lysyl Oxidase and Lysyl Oxidase-Like Enzymes
The Extracellular Matrix: an Overview, 2010Co-Authors: Herbert M. Kagan, Faina RyvkinAbstract:Lysyl Oxidase (LOX) and its four congeners, Lysyl Oxidase-like 1 (LOXL1), -2, -3, and -4, have received much investigative attention in recent years. LOX itself, is the prototypic form of these amine Oxidase enzymes. LOX has long been considered to function exclusively as the enzyme that oxidizes peptidyl lysine in its collagen and elastin substrates, thereby initiating formation of the covalent cross-linkages that stabilize these fibrous proteins. This view has been greatly expanded in light of the revelations that LOX can function both as an anti-oncogenic agent and as an enhancer of malignancy in selected cancerous conditions. Evidence is also accumulating that points to the roles of specific LOXL members of this family in disease and in biological homeostasis. This chapter reviews structural and catalytic properties as well as the roles in biology of these amine Oxidases and presents a computer-generated predicted 3D protein structure of LOX.
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Induction of human monocyte motility by Lysyl Oxidase
Matrix biology : journal of the International Society for Matrix Biology, 1995Co-Authors: H.m. Lazarus, William W. Cruikshank, N. Narasimhan, Herbert M. KaganAbstract:Lysyl Oxidase highly purified from calf aorta was found to be a potent chemotactic agent for unstimulated human peripheral blood mononuclear cells, determined in in vitro assays in Boyden chambers. A typical chemotactic bell-shaped curve was observed, with a maximal migratory response of 237% of control occurring at 10(-10) M Lysyl Oxidase. The chemotactic response was prevented by prior heat inactivation of the enzyme, by treatment of the enzyme with beta-aminopropionitrile or ethylenediamine, which are active site-directed inhibitors of Lysyl Oxidase, and by a competing, lysine-containing peptide substrate of Lysyl Oxidase. The chemoattractant response to Lysyl Oxidases was characterized by both chemokinetic and chemotactic components. These results raise the possibility that extracellular Lysyl Oxidase may have important roles to play in biology in addition to its established function in the crosslinking of elastin and collagen.
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Downregulation of Lysyl Oxidase in cadmium-resistant fibroblasts.
American journal of respiratory cell and molecular biology, 1995Co-Authors: Iih-nan Chou, Andra M. Boak, Herbert M. KaganAbstract:Lysyl Oxidase, a copper-dependent metalloenzyme, plays a central role in crosslinking of collagen and elastin in the extracellular matrix. Notably, lung Lysyl Oxidase activity is markedly stimulated in rats exposed to cadmium vapors. To further understand the mechanism of cadmium toxicity, the mRNA expression, synthesis, post-translational processing, and catalytic activity of Lysyl Oxidase were examined in cadmium-resistant (CdR) cells and the cadmium-sensitive Swiss mouse 3T3 cells from which they were derived. These CdR cells synthesized and accumulated markedly elevated levels of metallothionein, a known marker for cadmium resistance, whereas the expression of Lysyl Oxidase was reduced considerably. In comparison to the parental, cadmium-sensitive cells, the suppression of enzyme production in the CdR cells was seen at the mRNA level, at the levels of intracellular proprotein production and mature enzyme secreted into the medium, and in terms of total enzyme activity in the culture. The presence of cu...
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Lysyl Oxidase: Mechanism, Regulation and Relationship to Liver Fibrosis
Pathology research and practice, 1994Co-Authors: Herbert M. KaganAbstract:Summary Lysyl Oxidase oxidizes peptidyl lysine in collagen and elastin substrates to residues of α-aminoadipic-δ-semialdehyde. The peptidyl aldehydes can then undergo spontaneous condensations with unreacted ∈-amino groups and with neighboring aldehyde functions, thus forming the covalent crosslinkages which convert elastin and collagen into insoluble fibers. The unique role of Lysyl Oxidase in the post-translational modification of these proteins qualifies this enzymatic reaction as a potentially pivotal site of biological and/or chemotherapeutic control of collagen fiber deposition. Recent advances in the study of the catalytic mechanism, in the development of active site inhibitors, and in the biosynthesis and regulation of this unusual catalyst are reviewed as are studies on the response of Lysyl Oxidase in fibrotic liver.
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Reaction of Lysyl Oxidase with trans-2-phenylcyclopropylamine.
The Journal of biological chemistry, 1993Co-Authors: M A Shah, Philip C Trackman, Paul M. Gallop, Herbert M. KaganAbstract:Abstract trans-2-Phenylcyclopropylamine hydrochloride (tranylcypromine; TCP) was found to be both an inhibitor and a substrate of Lysyl Oxidase, the enzyme which oxidizes peptidyl lysine in elastin and collagen to initiate cross-linking in these proteins. The reaction of TCP with this enzyme was further characterized in view of the potential interference that chronic administration of this antidepressant compound may exert on the development and repair of connective tissues. In contrast to the irreversible and/or competitive inhibitors of Lysyl Oxidase previously described, TCP noncompetitively and reversibly inhibited the oxidation of both alkylamine and elastin substrates with Ki values of 386 and 375 microM, respectively. The noncompetitive mode of interaction affected the accessibility of the active site to productive amine substrates since the reductive trapping of n-hexylamine to Lysyl Oxidase was largely prevented by the presence of TCP. It was of additional interest that Lysyl Oxidase catalyzed a limited degree of conversion of TCP to cinnamaldehyde accompanied by the production of hydrogen peroxide. The lack of significant incorporation of protein-bound tritium accompanying reduction of the enzyme-TCP complex with [3H]NaBH4 argued against the formation of a Schiff base between the enzyme and the cinnamaldehyde product as the basis of the inhibitory effect. Spectral evidence was also obtained for an additional interaction between TCP and Lysyl Oxidase that was independent of the inhibitory effect of TCP. Cyclopropylamine, lacking the benzene moiety of TCP, inhibited Lysyl Oxidase irreversibly and competitively, and was not a substrate, pointing toward a defining role for the benzene moiety in the interaction of TCP with Lysyl Oxidase.
Amitha H Palamakumbura - One of the best experts on this subject based on the ideXlab platform.
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The Propeptide Domain of Lysyl Oxidase Induces Phenotypic Reversion of Ras-transformed Cells
The Journal of biological chemistry, 2004Co-Authors: Amitha H Palamakumbura, Ying Guo, Gail E. Sonenshein, P. Sommer, S. Jeay, N. Pischon, Philip C TrackmanAbstract:Abstract Lysyl Oxidase is an extracellular enzyme critical for the normal biosynthesis of collagens and elastin. In addition, Lysyl Oxidase reverts ras-mediated transformation, and Lysyl Oxidase expression is down-regulated in human cancers. Since suramin inhibits growth factor signaling pathways and induces Lysyl Oxidase in ras-transformed NIH3T3 cells (RS485 cells), we sought to investigate the effects of suramin on the phenotype of transformed cells and the role of Lysyl Oxidase in mediating these effects. Suramin treatment resulted in a more normal phenotype as judged by growth rate, cell cycle parameters, and morphology. β-aminopropionitrile, the selective inhibitor of Lysyl Oxidase enzyme activity, was remarkably unable to block suramin-induced reversion. By contrast, ectopic antisense Lysyl Oxidase demonstrated that Lysyl Oxidase gene expression mediated phenotypic reversion. Since Lysyl Oxidase is synthesized as a 50 kDa precursor and processed to a 30 kDa active enzyme and 18 kDa propeptide, the effects of these two products on the transformed phenotype of RS485 cells were then directly assessed in the absence of suramin. Here we report, for the first time, that the Lysyl Oxidase propeptide, and not the Lysyl Oxidase enzyme, inhibits ras-dependent transformation as determined by effects on cell proliferation assays, growth in soft agar, and Akt-dependent induction of NF-κB activity. Thus, the Lysyl Oxidase propeptide, which is released during extracellular proteolytic processing of pro-Lysyl Oxidase, functions to inhibit ras-dependent cell transformation.
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Autocrine growth factor regulation of Lysyl Oxidase expression in transformed fibroblasts.
The Journal of biological chemistry, 2003Co-Authors: Amitha H Palamakumbura, P. Sommer, Philip C TrackmanAbstract:Lysyl Oxidase catalyzes oxidative deamination of peptidyl-lysine and hydroxylysine residues in collagens and lysine residues in elastin to form peptidyl aldehydes that are required for the formation of covalent cross-links in normal extracellular matrix biosynthesis. Lysyl Oxidase in addition has tumor suppressor activity, and phenotypic reversion of transformed cell lines is accompanied by increased Lysyl Oxidase expression. The mechanism of low expression of Lysyl Oxidase in tumor cells is unknown. The present study investigates the hypothesis that autocrine growth factor pathways maintain low Lysyl Oxidase expression levels in c-H-ras-transformed fibroblasts (RS485 cell line). Autocrine pathways were blocked with suramin, a general inhibitor of growth factor receptor binding, and resulted in more than a 10-fold increase in Lysyl Oxidase expression and proenzyme production. This regulation was found to be reversible and occurred at the transcriptional level determined using Lysyl Oxidase promoter/reporter gene assays. Function blocking anti-fibroblast growth factor-2 (FGF-2) antibody enhanced Lysyl Oxidase expression in the absence of suramin. Finally, the addition of FGF-2 to suramin-treated cells completely reversed suramin stimulation of Lysyl Oxidase mRNA levels. Data support that an FGF-2 autocrine pathway inhibits Lysyl Oxidase transcription in the tumorigenic-transformed RS485 cell line. This finding may be of therapeutic significance and, in addition, provides a new experimental approach to investigate the mechanism of the tumor suppressor activity of Lysyl Oxidase.
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A fluorometric assay for detection of Lysyl Oxidase enzyme activity in biological samples.
Analytical biochemistry, 2002Co-Authors: Amitha H Palamakumbura, Philip C TrackmanAbstract:Abstract Lysyl Oxidase catalyzes the final known enzymatic step required for collagen and elastin cross-linking in the biosynthesis of normal mature functional insoluble extracellular matrices. In addition, Lysyl Oxidase has been identified as a possible tumor suppressor. Lysyl Oxidase activity in biological samples is traditionally and most reliably assessed by tritium release end-point assays using radiolabeled collagen or elastin substrates involving laborious vacuum distillation of the released tritiated water. In addition, a less sensitive fluorometric method exists that employs nonpeptidyl amine Lysyl Oxidase substrates and measures hydrogen peroxide production with horseradish perOxidase coupled to homovanillate oxidation. The present study describes a more sensitive fluorescent assay for Lysyl Oxidase activity that utilizes 1,5-diaminopentane as substrate, and released hydrogen peroxide is detected using Amplex red in horseradish perOxidase-coupled reactions. This method allows the detection of 40 ng of enzyme per 2 ml assay at 37°C and is 7.5 times more sensitive than the currently available fluorometric assay for enzyme activity. This method eliminates the interference that occurs in some biological samples and can be successfully used to detect Lysyl Oxidase activity in cell culture experiments.
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Multiple Bone Morphogenetic Protein 1-related Mammalian Metalloproteinases Process Pro-Lysyl Oxidase at the Correct Physiological Site and Control Lysyl Oxidase Activation in Mouse Embryo Fibroblast Cultures
Journal of Biological Chemistry, 2001Co-Authors: Mehmet Ilhan Uzel, Daniel S. Greenspan, Amitha H Palamakumbura, William N Pappano, Hermik Babakhanlou-chase, Hsiang-hsi Hong, Ian C. Scott, Philip C TrackmanAbstract:Abstract Lysyl Oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-Lysyl Oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I–III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-Lysyl Oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-Lysyl Oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in Lysyl Oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null,Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for Lysyl Oxidase enzyme activity and for accumulation of pro-Lysyl Oxidase and mature ∼30-kDa Lysyl Oxidase. Wild type cells or cells singly null for Bmp1 orTll1 all produced both pro-Lysyl Oxidase and processed Lysyl Oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-Lysyl Oxidase and had Lysyl Oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, andTll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of Lysyl Oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-Lysyl Oxidase as the first known substrate for mTLL-2.
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Multiple Bone Morphogenetic Protein 1-related Mammalian Metalloproteinases Process Pro-Lysyl Oxidase at the Correct Physiological Site and Control Lysyl Oxidase Activation in Mouse Embryo Fibroblast Cultures
The Journal of biological chemistry, 2001Co-Authors: Mehmet Ilhan Uzel, Daniel S. Greenspan, Amitha H Palamakumbura, William N Pappano, Hermik Babakhanlou-chase, Hsiang-hsi Hong, Ian C. Scott, Philip C TrackmanAbstract:Lysyl Oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-Lysyl Oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-Lysyl Oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-Lysyl Oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in Lysyl Oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for Lysyl Oxidase enzyme activity and for accumulation of pro-Lysyl Oxidase and mature approximately 30-kDa Lysyl Oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-Lysyl Oxidase and processed Lysyl Oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-Lysyl Oxidase and had Lysyl Oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of Lysyl Oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-Lysyl Oxidase as the first known substrate for mTLL-2.
Mehmet Ilhan Uzel - One of the best experts on this subject based on the ideXlab platform.
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Multiple Bone Morphogenetic Protein 1-related Mammalian Metalloproteinases Process Pro-Lysyl Oxidase at the Correct Physiological Site and Control Lysyl Oxidase Activation in Mouse Embryo Fibroblast Cultures
The Journal of biological chemistry, 2001Co-Authors: Mehmet Ilhan Uzel, Daniel S. Greenspan, Amitha H Palamakumbura, William N Pappano, Hermik Babakhanlou-chase, Hsiang-hsi Hong, Ian C. Scott, Philip C TrackmanAbstract:Lysyl Oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-Lysyl Oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-Lysyl Oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-Lysyl Oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in Lysyl Oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for Lysyl Oxidase enzyme activity and for accumulation of pro-Lysyl Oxidase and mature approximately 30-kDa Lysyl Oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-Lysyl Oxidase and processed Lysyl Oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-Lysyl Oxidase and had Lysyl Oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of Lysyl Oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-Lysyl Oxidase as the first known substrate for mTLL-2.
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Multiple Bone Morphogenetic Protein 1-related Mammalian Metalloproteinases Process Pro-Lysyl Oxidase at the Correct Physiological Site and Control Lysyl Oxidase Activation in Mouse Embryo Fibroblast Cultures
Journal of Biological Chemistry, 2001Co-Authors: Mehmet Ilhan Uzel, Daniel S. Greenspan, Amitha H Palamakumbura, William N Pappano, Hermik Babakhanlou-chase, Hsiang-hsi Hong, Ian C. Scott, Philip C TrackmanAbstract:Abstract Lysyl Oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-Lysyl Oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I–III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-Lysyl Oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-Lysyl Oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in Lysyl Oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null,Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for Lysyl Oxidase enzyme activity and for accumulation of pro-Lysyl Oxidase and mature ∼30-kDa Lysyl Oxidase. Wild type cells or cells singly null for Bmp1 orTll1 all produced both pro-Lysyl Oxidase and processed Lysyl Oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-Lysyl Oxidase and had Lysyl Oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, andTll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of Lysyl Oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-Lysyl Oxidase as the first known substrate for mTLL-2.
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Molecular events that contribute to Lysyl Oxidase enzyme activity and insoluble collagen accumulation in osteosarcoma cell clones.
Journal of Bone and Mineral Research, 2000Co-Authors: Mehmet Ilhan Uzel, Sarah D. Shih, Howard Gross, Efrat Kessler, Louis C Gerstenfeld, Philip C TrackmanAbstract:Maximum collagen synthesis and maximum accumulation of insoluble collagen occur at different phenotypic stages in developing osteoblastic cell cultures. Insoluble collagen accumulation depends in part on the activity of extracellular enzymes including procollagen N-proteinases, procollagen C-proteinase (derived from the BMP1 gene), and Lysyl Oxidase. In addition to its action on procollagen, procollagen C-proteinase processes proLysyl Oxidase to mature 32-kDa Lysyl Oxidase. The regulation of extracellular activities that control insoluble collagen accumulation has not been studied extensively. The present study compares molecular events that control production of a collagenous mineralized extracellular matrix in vitro among five different murine osteosarcoma cell clones derived from the same tumor, but which differ in their ability to produce an insoluble mineralized matrix. Levels of insoluble type I collagen, insoluble calcium, bone morphogenetic protein 1 (BMP-1), and Lysyl Oxidase expression, Lysyl Oxidase biosynthesis, Lysyl Oxidase activity, and proLysyl Oxidase processing activity were determined. Results surprisingly indicate that Lysyl Oxidase activity is not related closely to Lysyl Oxidase messenger RNA (mRNA) levels among the different cell clones. However, it appears that BMP-1-dependent proLysyl Oxidase processing could contribute to the observed Lysyl Oxidase activity. Highest collagen and BMP-1 mRNA levels, proLysyl Oxidase processing activity, and Lysyl Oxidase activity occurred in a cell clone (K8) that showed the highest levels of insoluble collagen accumulation. Culture media from a cell clone (K37) that accumulates little insoluble collagen or calcium but expresses high levels of Lysyl Oxidase mRNA contained low molecular weight fragments of Lysyl Oxidase protein and showed low Lysyl Oxidase activity. By contrast the K14 cell line exhibits relatively high Lysyl Oxidase activity and collagen accumulation, but low levels of mature Lysyl Oxidase protein. Together, these studies indicate that catabolic as well as anabolic activities are important in regulating insoluble collagen accumulation in osteoblastic cells. In addition, results suggest that products of genes homologous to Lysyl Oxidase may contribute to observed Lysyl Oxidase activity.
Hsiang-hsi Hong - One of the best experts on this subject based on the ideXlab platform.
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Cytokine Regulation of Gingival Fibroblast Lysyl Oxidase, Collagen, and Elastin
Journal of periodontology, 2002Co-Authors: Hsiang-hsi Hong, Philip C TrackmanAbstract:Background: Systemic therapy with cyclosporin A, phenytoin, and nifedipine modulates cytokine levels in human gingival tissues. Functional relationships between altered cytokine levels and gingival extracellular matrix production are partially characterized. The present study investigates in cultured human gingival fibroblasts the regulation of Lysyl Oxidase, α-1 type I collagen, and elastin by selected cytokines that are elevated in drug-induced gingival overgrowth tissues. Methods: Normal human gingival fibroblasts were cultured and then treated with selected cytokines: interleukin (IL)-1β, IL- 6, platelet-derived growth factor (PDGF)-BB, and basic fibroblast growth factor (bFGF or FGF-2). Cells were harvested at intervals, and changes in Lysyl Oxidase enzyme activity, and in mRNA levels of Lysyl Oxidase, α-1 type I collagen, and elastin were determined. Results: bFGF reproducibly and significantly decreased human gingival fibroblast Lysyl Oxidase and α-1 type I collagen mRNA levels in a dose- and time-...
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Multiple Bone Morphogenetic Protein 1-related Mammalian Metalloproteinases Process Pro-Lysyl Oxidase at the Correct Physiological Site and Control Lysyl Oxidase Activation in Mouse Embryo Fibroblast Cultures
Journal of Biological Chemistry, 2001Co-Authors: Mehmet Ilhan Uzel, Daniel S. Greenspan, Amitha H Palamakumbura, William N Pappano, Hermik Babakhanlou-chase, Hsiang-hsi Hong, Ian C. Scott, Philip C TrackmanAbstract:Abstract Lysyl Oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-Lysyl Oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I–III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-Lysyl Oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-Lysyl Oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in Lysyl Oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null,Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for Lysyl Oxidase enzyme activity and for accumulation of pro-Lysyl Oxidase and mature ∼30-kDa Lysyl Oxidase. Wild type cells or cells singly null for Bmp1 orTll1 all produced both pro-Lysyl Oxidase and processed Lysyl Oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-Lysyl Oxidase and had Lysyl Oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, andTll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of Lysyl Oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-Lysyl Oxidase as the first known substrate for mTLL-2.
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Multiple Bone Morphogenetic Protein 1-related Mammalian Metalloproteinases Process Pro-Lysyl Oxidase at the Correct Physiological Site and Control Lysyl Oxidase Activation in Mouse Embryo Fibroblast Cultures
The Journal of biological chemistry, 2001Co-Authors: Mehmet Ilhan Uzel, Daniel S. Greenspan, Amitha H Palamakumbura, William N Pappano, Hermik Babakhanlou-chase, Hsiang-hsi Hong, Ian C. Scott, Philip C TrackmanAbstract:Lysyl Oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-Lysyl Oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-Lysyl Oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-Lysyl Oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in Lysyl Oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for Lysyl Oxidase enzyme activity and for accumulation of pro-Lysyl Oxidase and mature approximately 30-kDa Lysyl Oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-Lysyl Oxidase and processed Lysyl Oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-Lysyl Oxidase and had Lysyl Oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of Lysyl Oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-Lysyl Oxidase as the first known substrate for mTLL-2.
