TRANSLATIONAL CONTROL OF INFLAMMATORY GENE EXPRESSION
Paul L Fox, Professor
Cleveland Clinic Lerner Col/med-cwru, Jjn5-01, Cleveland, Oh 44195
Abstract: The long-term goal of Project 2 is to elucidate the post-transcriptional mechanisms that modulate gene expression in inflammation of the vasculature. Interferon (IFN)-v is the classic activator of monocyte/macro- phages, and it induces rapid transcription of inflammatory growth factors, proteases, chemokines, and gen- erators of radical species. If unregulated, this process becomes chronic and monocyte/macrophage products accumulate, damage host tissue, and contribute to chronic disorders of blood vessels, e.g., atherosclerosis. The termination of inflammation is not a passive process that begins after elimination of the initial insult; in contrast, intrinsic mechanisms actively limit expression of potentially injurious proteins. Recently, investigat- ors have recognized the important role of post-transcriptional processes in limiting or resolving inflammation. We have discovered a novel translational control pathway that acts as an endogenous regulator of the inflammatory response. In myeloid cells, IFN-y induces assembly of the heterotetrameric, IFN-Gamma- Activated inhibitor of Translation (GAIT) complex, which binds an RNA element in the 3´untranslated region of certain pro-inflammatory target mRNAs, e.g., vascular endothelial growth factor-A, and inhibits their translation. In Preliminary Studies we show that one GAIT protein, glutamyl-prolyl-tRNA synthetase (EPRS), is central to the GAIT system because it is responsible for target mRNA recognition, and its function is regu- lated by phosphorylation and binding of the other 3 GAIT proteins. We suggest EPRS is not an inert, protein- binding scaffold, but rather a dynamic system subject to stimulus-inducible modifications that regulate GAIT complex assembly and function. Based on these results, we propose the following hypothesis Phosphoryl- ation of EPRS by IFN-y-dependent kinases causes conformational changes in EPRS that regulate assembly of the GAIT complex, which silences translation of inflammatory mRNA targets and contributes to the resolu- tion of chronic inflammation. We will test this hypothesis by pursuit of three Specific Aims. In Aim 1 we will determine the EPRS domains required for GAIT complex assembly and GAIT element-binding. In Aim 2 we will determine the role of EPRS phosphorylation in GAIT complex assembly and function. In Aim 3 we will investigate the anti-inflammatory function of EPRS and the GAIT complex in vivo. RELEVANCE (See instructions) Our studies will elucidate a new pathway that regulates the synthesis of inflammatory proteins by macrophages, an important process in the development of vascular diseases such as atherosclerosis. The pathway under investigation contributes to the limitation and resolution of chronic inflammation, an important causative factor in disease progression. A deeper understanding of inflammatory "stop" pathways is important because defects in these pathways can contribute to vascular disorders, and because the pathway itself may present alternative targets for development of novel anti- inflammatory therapeutics
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
5P01HL029582-27_8735 (2010): $261119
Sponsored Links Excellgen http://Excellgen.com
MACROMOLECULAR INTERACTION CORE
Paul L Fox, Professor
Cleveland Clinic Lerner Col/med-cwru, Jjn5-01, Cleveland, Oh 44195
Abstract: The scope of the Atherosclerosis and Lipoprotein Analysis Core facility is the quantitative assessment of atherosclerosis lesion areas at two locations in mouse models. For the aortic root assay, each individual investigator is only responsible for providing the excised saline-perfused heart in 10% phosphate buffered formalin. The Core personnel will then 1) embed the heart in gelatin, 2) freeze the gelatin block in OCT, 3) prepare 5 slides containing four 12.5 micron thick sections each, that cover the first 500 microns of the aortic root, 4) stain the slides in oil red O, hematoxylin, and fast green, and 5) quantitate the lesion areas on one section per slide and calculate the mean lesion are per section. For the en face assay, the Core will provide a trained technologist to work under the project leader´s approved protocol to 1) anesthetize the mice, 2) perform saline perfusion, which euthanizes the mouse by exsanguination, 3) dissect the mouse to display the mouse aorta in situ, 4) remove the aorta and trim off adventitial tissue, 5) cut the aorta longitudinally and fix it flattened between two microscope slides, 6) stain the aorta with oil red O, 7) mount the aorta on a microscope slide, and 8) quantify the % surface lesion area in the entire aorta, as well as in the regions of the aortic arch, and thoracic and abdominal aortas
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
5P01HL029582-27_8740 (2010): $261119
CERULOPLASMIN OXIDANT ACTIVITY IN ATHEROSCLEROSIS
Paul L Fox
Cleveland Clinic Foundation 9500 Euclid Ave. Cleveland, Oh 44195
Grant 5P01HL029582-200009 from National Heart, Lung, And Blood Institute
Abstract: Oxidative modification of low density lipoprotein (LDL) is thought to be an important event during atherogenesis. The mechanism(s) by which lipoproteins are oxidized in vivo is largely unknown and a major-long term goal of this project. During the last several years we have explored the pro-oxidant activity of ceruloplasmin, a Cu-containing plasma protein, and have shown that it plays an important role in monocytic cell-mediated oxidative processes. Our evidence is that purified human ceruloplasmin induces LDL oxidation in vitro, that cultured monocytic cell-mediated oxidative processes. Our evidence is that purified human ceruloplasmin induces LDL oxidation in vitro, that cultured monocytic cells secrete ceruloplasmin is regulated at the mRNA and translational levels, and that immunohistochemical analysis shows that ceruloplasmin, in association with macrophages, is abundant in atherosclerotic lesions of human carotid endarterectomy specimens. These findings have led us to propose the following hypothesis That lesion-derived cytokines stimulate macrophage production of ceruloplasmin, which as a Cu-containing, pro-oxidant molecule accelerates lipoprotein oxidation and atherosclerotic lesion formation. We will test this hypothesis by pursuing three specific aims. We will use biochemical and molecular approaches to determine the domains of ceruloplasmin involved in oxidant activity. We will investigate transcriptional and translational mechanisms that regulate ceruloplasmin synthesis by monocytic cells. Finally, we will determine the role of ceruloplasmin in vessel wall lipoprotein oxidation and atherosclerotic lesion formation in vivo. To accomplish this last aim, we will generate transgenic mice that over express ceruloplasmin and breed them with atherosclerosis-susceptible apo E-deficient mice. An understanding of the mechanisms underlying vessel wall oxidation processes is likely to have a major impact on the development of treatments that specifically reduce pathological oxidation while minimizing the effect of normal oxidative processes.
Keywords: atherosclerosis, blood lipoprotein metabolism, ferroxidase, low density lipoprotein, molecular pathology, oxidation, apolipoprotein E, oxidative stress, oxidized lipid, laboratory mouse, transgenic animal
Grants awarded to Paul L Fox
OXIDANT ROLE OF CERULOPLASMIN IN ATHEROSCLEROSIS
Paul L Fox, Professor Of Molecular Medicine
Cleveland Clinic Foundation 9500 Euclid Ave. Cleveland, Oh 44195
Grant 5R01HL052692-03 from National Heart, Lung, And Blood Institute IRG: HEM
Abstract: The regulation of oxidant and antioxidant activity has special significance in the development of cardiovascular disease in general, and atherosclerosis in particular. Recent evidence suggests that low density lipoprotein (LDL) that is modified by oxidation accumulates in atherosclerotic lesions, and in vitro studies suggest that oxidized LDL may be responsible for the dysfunctional behavior of lesion cells. The mechanisms of LDL oxidation are not known, but the cells present in lesions can oxidize LDL in vitro. The role of ceruloplasmin in any of these processes has not been investigated, but numerous reports of a potent antioxidant activity that can block the oxidation of lipids motivated us to begin studies of its role in LDL oxidation and lesion formation. Ceruloplasmin is an abundant 132 kDa acute-phase copper-protein carrying 95% of the copper in plasma. In contrast to previous reports, our preliminary studies show that ceruloplasmin is an extremely potent oxidant; at physiological levels it increases the oxidation of LDL by at least 25-fold. We have found that ceruloplasmin function is extremely sensitive to structural modification since oxidant activity (but not a distinct oxidase activity) is completely suppressed by (1) the removal of a single, specific bound copper or by (2) a single proteolytic event that cleaves ceruloplasmin into 116 and 19 kDa fragments. The latter finding may explain earlier reports of antioxidant activity. In other preliminary studies, we have observed that ceruloplasmin may be involved in cell- mediated oxidation. Ceruloplasmin can substitute for free metal ions in stimulating LDL oxidation by endothelial cells and smooth muscle cells. Ceruloplasmin may also regulate macrophage oxidation of LDL; in preliminary studies, we have found that agents that stimulate LDL oxidation by activated U937 cells (a monocytic line) also stimulate ceruloplasmin gene expression and protein production. Furthermore, anti- ceruloplasmin antibodies suppress much of the oxidant activity of U937 cells. Finally, a role for ceruloplasmin in oxidative processes in lesions is supported by our recent observation that ceruloplasmin is abundant in atherosclerotic lesions, but not in adjacent non-lesioned areas, of human carotid endarterectomy specimens. These new findings have led us to propose that the induced synthesis and secretion of ceruloplasmin by activated monocytes contributes to the oxidation of LDL by these and other vascular cells, and thus plays a critical role in pathological accumulation of oxidized lipoproteins in the vessel wall. The hypothesis will be tested in this proposal by examining the structural features of ceruloplasmin required for oxidant activity, by investigating its interaction with LDL, by determining the role of ceruloplasmin in vascular cell-mediated oxidation of LDL, and finally, by studies on the distribution and activity of ceruloplasmin in atherosclerotic lesions.
Keywords: atherosclerosis, enzyme activity, enzyme mechanism, ferroxidase, low density lipoprotein, oxidation, antioxidant, chemical binding, copper, enzyme biosynthesis, enzyme structure, histidine, molecular site, monocyte, oxidizing agent, vascular endothelium, immunocytochemistry, site directed mutagenesis, tissue /cell culture
Project start date: 1994-12-01
Project end date: 1998-11-30
5R01HL052692-03 (1997): $205261
Translational Control Of Inflammatory Gene Expression
Paul L Fox, Professor Of Molecular Medicine
Molecular Medicinecleveland Clinic Lerner Col/med-cwru
Grant 1R01HL094441-01 from National Heart, Lung, And Blood Institute IRG: VCMB
Abstract: The long-term goal of our research program is to elucidate the post-transcriptional mechanisms that modulate gene expression in inflammation of the vasculature. Interferon (IFN)-3 is the classic activator of monocyte/macrophages, and it induces rapid transcription of inflammatory growth factors, proteases, chemokines, and generators of radical species. If unregulated, this process becomes chronic and monocyte/macrophage products accumulate, damage host tissue, and contribute to chronic disorders of blood vessels, e.g., atherosclerosis. The termination of inflammation is not a passive process that begins after elimination of the initial insult; in contrast, intrinsic mechanisms actively limit expression of potentially injurious proteins. Recently, investigators have recognized the important role of post-transcriptional processes in limiting or resolving inflammation. We have discovered a novel translational control pathway that acts as an endogenous regulator of the inflammatory response. In myeloid cells, IFN-3 induces assembly of the heterotetrameric, IFN-Gamma-Activated Inhibitor of Translation (GAIT) complex, which binds an RNA element in the 3´untranslated region of certain pro-inflammatory target mRNAs, e.g., vascular endothelial growth factor-A, and inhibits their translation. In Preliminary Studies we show that one GAIT protein, glutamyl-prolyl-tRNA synthetase (EPRS), is central to the GAIT system because it is responsible for target mRNA recognition, and its function is regulated by phosphorylation and binding of the other 3 GAIT proteins. We suggest EPRS is not an inert, protein-binding scaffold, but rather a dynamic system subject to stimulus-inducible modifications that regulate GAIT complex assembly and function. Based on these results, we propose the following hypothesis Phosphorylation of EPRS by IFN-3- dependent kinases causes conformational changes in EPRS that regulate assembly of the GAIT complex, which silences translation of inflammatory mRNA targets and contributes to the resolution of chronic inflammation. We will test this hypothesis by pursuit of three Specific Aims. In Aim 1 we will determine the EPRS domains required for GAIT complex assembly and GAIT element-binding. In Aim 2 we will determine the role of EPRS phosphorylation in GAIT complex assembly and function. In Aim 3 we will investigate the anti- inflammatory function of EPRS and the GAIT complex in vivo. Dysregulation of anti-inflammatory mechanisms in vascular disease is an important, under-investigated area. Our studies will elucidate the molecular mechanism of a post-transcriptional pathway that regulates the protein expression profile of inflammatory macrophages, and will improve our understanding of mechanisms in the limitation and resolution of chronic inflammation. Understanding endogenous, anti-inflammatory "off" mechanisms is crucial because defects can contribute to inflammatory disorders, and because the pathway itself may present alternative targets for development of novel anti-inflammatory therapeutics to reduce vascular disease. Our studies will elucidate a new pathway that regulates the synthesis of inflammatory proteins by macrophages, an important process in the development of vascular diseases such as atherosclerosis. The pathway under investigation contributes to the limitation and resolution of chronic inflammation, an important causative factor in disease progression. A deeper understanding of inflammatory "stop" pathways is important because defects in these pathways can contribute to vascular disorders, and because the pathway itself may present alternative targets for development of novel anti-inflammatory therapeutics
Project start date: 2008-12-01
Project end date: 2013-11-30
MARINE LIPIDS AND ENDOTHELIAL CELLS IN ATHEROSCLEROSIS
Paul L Fox, Professor Of Molecular Medicine
Cleveland Clinic Foundation 9500 Euclid Ave. Cleveland, Oh 44195
Grant 5R29HL040352-05 from National Heart, Lung, And Blood Institute IRG: PTHA
Abstract: The long term objective of this proposal is to understand the role of marine lipids on lesion formation in atherosclerosis. The proposal is based on four observations (1) that smooth muscle cell (SMC) proliferation is an early and critical event in the formation of occlusive atherosclerotic lesions; (2) that ingestion of fish or fish oils containing n-3 polyunsaturated fatty acids (PUFAs) inhibits intimal thickening in experimental animals, and may decrease the incidence of coronary heart disease in man; (3) that cultured endothelial cells (EC) produce mitogens, especially a platelet-derived growth factor-like protein, that stimulate SMC proliferation in vitro; and (4) that growth factor production by cultured EC is regulatable and, as we have recently observed, is inhibited by fish oil emulsions and certain modified lipoproteins. To explain these observations, we propose that ingestion of fish oils results in an elevated level of specific marine lipids in plasma lipoproteins, that the altered lipoproteins inhibit EC production of growth factors, and that this regulatory mechanism is in part responsible for the observed decrease in SMC growth, and the proposed benefits of this diet. This hypothesis will be tested by pursuit of the following specific aims 1. To characterize and identify the lipid(s) in marine oils that specifically inhibits EC production of growth factors. 2. To determine if cellular processing is required for the expression of the inhibitory activity of marine lipids. 3. To determine the biosynthetic step(s) regulated by marine lipids that causes the inhibition of growth factor production by EC. 4. To determine whether physiological lipoproteins enriched with marine lipids inhibit EC production of growth factors. 5. To determine if marine lipids inhibit the proliferation of cultured vascular SMC. Answers to these questions of vascular cell biology should contribute to our understanding of the cellular interactions of EC and SMC. The mechanistic insights resulting from this work may help to explain the recent epidemiological studies which suggest that diets rich in n-3 PUFAs are associated with reduced incidence of coronary heart disease, and may eventually lead to the development of pharmacologic inhibitors of atherogenesis.
Keywords: atherosclerosis, chemoprevention, growth inhibitor, marine animal oil, omega 3 fatty acid, platelet derived growth factor, vascular endothelium, blood lipoprotein metabolism, cardiovascular disorder chemotherapy, cardiovascular disorder prevention, coronary disorder, diet therapy, fatty acid biosynthesis, nonnutrition disorder nutrition therapy, vascular smooth muscle, high performance liquid chromatography, human tissue, mixed tissue /cell culture, nucleic acid probe, nutrition related tag, radiotracer, thin layer chromatography
Project start date: 1988-04-01
Project end date: 1995-03-31
5R29HL040352-05 (1992): $93695
Macrophage Ceruloplasmin Expression In Atherosclerosis
Paul L Fox, Professor Of Molecular Medicine
Cleveland Clinic Foundation 9500 Euclid Ave. Cleveland, Oh 44195
Grant 2P01HL029582-210003 from National Heart, Lung, And Blood Institute IRG: ZHL1
Abstract: Our long-term goal is to understand the role of ceruloplasmin (Cp) in normal and pathological vascular processes, particularly atherosclerosis. Cp is an acute phase, inflammatory protein produced by the liver and by activated monocyte/ macrophages. Clinical studies indicate that elevated serum Cp is an important risk factor for atherosclerosis and other cardiovascular diseases. Cp is a multifunctional enzyme with several activities that may contribute to pathological vascular processes including LDL oxidase activity due to a surface copper and a ferroxidase activity that regulates tissue iron homeostasis. Besides the constitutive, systemic production by hepatocytes, Cp is secreted locally by mac-rophages in inflammatory sites, including in human atherosclerotic lesions. We have shown that interferon (IFN)-gamma, a contributor to lesion progression in murine models of atherosclerosis, stimulates Cp mRNA and protein expression by myeloid cells, including U937 cells and peripheral blood monocytes. Cp synthesis abruptly stops about 16 h after IFN-gamma treatment, despite the continued presence of high levels of Cp mRNA. We have described a novel translational silencing mechanism for this inhibition of Cp expression. Silencing depends on end-to-end circularization of the Cp transcript via interaction of the Cp 3 -terminus with the 5 -translation-initiation complex. In new preliminary studies we show that silencing is driven by a 29-nt element in the Cp 3 -untranslated region (UTR). We also show that cytosolic extracts from IFN-gamma-treated U937 cells contain a protein or complex, denoted IFN-Gamma-Activated Inhibitor of Translation (GAIT), that binds to the 29-nt GAIT element (GAITE) and blocks Cp translation in vitro. By RNA/protein interaction- cloning with the yeast 3-hybrid system we have identified candidate proteins that specifically bind to the GAIT element and block Cp translation in vitro. These results have led us to propose the following hypothesis IFN-gamma-stimulated production of Cp by macrophages is a critical contributor to atherogenesis. In specific, IFN-gamma induces myeloid Cp synthesis, but it also causes a delayed activation or induction of cellular regulatory proteins that bind the Cp 3 -UTR GAITE, and subsequently silence translation of Cp (and possibly other related proteins). Dysregulation of this silencing mechanism may lead to prolonged production of Cp, accumulation of Cp to injurious levels, and accelerated atherosclerotic lesion progression. We will test this hypothesis by pursuing the following Specific Aims (1) identify Cp 3 -UTR-binding proteins and determine their function in translational silencing of Cp by IFN-gamma, (2) determine the cellular function of translational silencing of Cp, and (3) determine the role of Cp in atherosclerotic lesion progression in genetically-altered mice. We believe that these studies will yield important information on the basic mechanisms of translational control relevant to IFN-gamma-activated macrophages, and in particular, on the endogenous mechanisms involved in the termination of inflammation. The studies will also help to elucidate the vascular function of macrophage-derived Cp and its role in atherosclerotic lesion progression.
Keywords: atherosclerosis, enzyme activity, ferroxidase, gene expression, macrophage, posttranslational modification, protein structure function, binding protein, gene induction /repression, genetic translation, interferon gamma, low density lipoprotein, monocyte, oxidation, protein protein interaction, affinity chromatography, clinical research, laboratory mouse, mass spectrometry, transgenic animal, yeast two hybrid system
Project start date: 2003-07-01
Project end date: 2008-06-30
A PROTEIN-DIRECTED RIBOSWITCH IN THE VEGF-A 3´UTR THAT REGULATES TRANSLATION
Paul L Fox, Professor
Cleveland Clinic Lerner Col/med-cwru, Jjn5-01, Cleveland, Oh 44195
Grant 5R01GM086430-02 from National Institute Of General Medical Sciences
Abstract: Our long-term goal is to understand the interactions between elements in noncoding regions of vertebrate mRNAs, and their cognate binding proteins, and how they integrate signals from disparate stimuli to control translation. Transcript-selective translational control is mediated by interactions of RNA-binding proteins to sequence/structural elements in non-coding regions, most often the 5´- or 3´-untranslated region (UTR) of the target transcript. In addition to protein-RNA interactions, RNA-RNA interactions also regulate gene expression, e.g., riboswitches in the UTR of bacterial mRNAs contain proximate structural elements that undergo conformational change in response to specific metabolites, and control translation. Recent experiments in our laboratory suggest that human vascular endothelial growth factor (VEGF)-A mRNA contains adjoining elements that function as a novel stimulus-dependent, protein-directed riboswitch that exists in two metastable conformations a translation-silencing and a translation-permissive conformer. The binary switch is controlled by integration of two signals, interferon (IFN)-? and hypoxia, that regulate the amount or activity of the binding factors. Upon cell stimulation by IFN-?, Glu-Pro tRNA synthetase (EPRS) is released from its residence from the tRNA multisynthetase complex and joins the GAIT (IFN-Gamma-Activated Inhibitor of Translation) complex. EPRS binds a defined, 29-nt GAIT element in the VEGF-A mRNA 3´UTR, stabilizing the translation-silencing conformer and inhibiting translation. However, superimposition of hypoxia on IFN-? stimulation increases the level of heterogeneous nuclear ribonucleoprotein (hnRNP) L that binds a CA-rich element directly upstream of the GAIT element, stabilizing the translation-permissive conformer and allowing VEGF-A expression. We propose the following specific hypothesis The myeloid cell integrates signals from IFN-? and hypoxia by regulating the relative amounts of hnRNP L and GAIT complex, which in turn dictate the conformation of the VEGF-A 3´UTR to either permit or suppress VEGF-A mRNA translation. We will test this hypothesis by pursuing the following Specific Aims Aim 1 Determine sequences and secondary structures in the VEGF-A mRNA 3´UTR required for binary switch function. Aim 2 Determine the role of VEGF-A 3´UTR binding proteins in switch function. Aim 3 Investigate regulation of the VEGF-A 3´UTR binary switch by IFN-? and hypoxia. We hypothesize that the switch evolved to maintain VEGF-A expression and angiogenesis in hypoxic, inflammatory tissues. Tumors, also residing in hypoxic, inflammatory sites, may take advantage of the VEGF-A switch to stimulate inward blood vessel growth to provide nourishment and permit tumor growth. Thus, the VEGF-A switch represents a novel therapeutic target to specifically inhibit tumor macrophage expression of VEGF-A. We also speculate that the VEGF-A switch may represent the founding member of a family of protein-directed riboswitches in vertebrates that integrate other physiological or pathological stimuli to control gene expression. PUBLIC HEALTH RELEVANCE Certain messenger RNAs respond to changes in their environment by altering their folding structure and their rate of expression of protein products. Although these "riboswitches" are found primarily in bacteria, we have found a similar switch in the mRNA encoding human vascular endothelial growth factor (VEGF), a protein critical for blood vessel formation. The VEGF riboswitch is sensitive to inflammation and hypoxia, two conditions found in the tumor environment, and an understanding of its mechanism may reveal insights into tumor growth and potential therapies to inhibit the process
Keywords: 20S Catalytic Proteasome; 20S Core Proteasome; 20S Proteasome; 20S Proteosome; 3` Untranslated Regions; 3`UTR; Affinity; Amino Acyl T RNA Synthetases; Amino Acyl-tRNA Ligases; Amino Acyl-tRNA Synthetases; Aminoacyl Transfer RNA Synthetase; Aminoacyl-tRNA Synthetase; Bacteria; Be element; Be++ element; Beryllium; Binding; Binding (Molecular Function); Binding Proteins; Binding Sites; Bio-Informatics; Bioinformatics; Blood Vessels; Body Tissues; Cell Communication and Signaling; Cell Signaling; Cells; Chronic; Combining Site; Complex; Deletion Mutation; Development; EPRS enzyme; Elements; Environment; Functional RNA; GFAC; Gamma interferon; Gene Expression; Gene Products, RNA; Generalized Growth; Genetic Translation; Goals; Growth; Growth Agents; Growth Factor; Growth Factors, Proteins; Growth Substances; HBGF; Heterogeneous-Nuclear Ribonucleoprotein L; Heterogeneous-Nuclear Ribonucleoproteins; Hypoxia; Hypoxic; IFN; IFN-Gamma; IFN-g; IFNG; INFLM; In element; Indium; Inflammation; Inflammatory; Informofer; Interferon Gamma; Interferon Type II; Interferon gamma (human lymphocyte protein moiety reduced); Interferon, Immune; Interferon-gamma; Interferons; Intracellular Communication and Signaling; Kinetic; Kinetics; Laboratories; Ligand Binding Protein; Macropain; Macroxyproteinase; Malignant Cell; Maps; Mediating; Messenger RNA; Methods; Micro RNA; MicroRNAs; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Multicatalytic Proteinase; Mutation Analysis; Myeloid Cells; Non-Coding; Non-Coding RNA; Nucleases, RNA; Oxygen Deficiency; Pathway interactions; Phosphorylation; Physiologic; Physiological; Plants; Plants, General; Pro-tRNA; Process; Production; Prosome; Proteasome; Proteasome Endopeptidase Complex; Protein Family; Protein Phosphorylation; Proteins; Proteosome; RNA; RNA, Messenger; RNA, Non-Polyadenylated; RNA-Binding Proteins; RNA-Protein Interaction; RNase; Reactive Site; Regulation; Relative; Relative (related person); Repression; Resolution; Ribonuclease Family Protein; Ribonucleases; Ribonucleic Acid; Ribonucleic acids, transfer; Role; Signal Transduction; Signal Transduction Systems; Signaling; Site; Stimulus; Structure; System; System, LOINC Axis 4; Testing; Tissue Growth; Tissues; Transcript; Transfer RNA; Transfer RNA Synthetase; Translations; Triplet Codon-Amino Acid Adaptor; Tumor Angiogenesis; UTRs; Untranslated Regions; VEGF protein, human; VEGF-A protein, human; VEGF148; VEGFA protein, human; VEGFs; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vasculotropin; Vegf; Vertebrate Animals; Vertebrates; aminoacid tRNA ligase; angiogenesis; base; biological signal transduction; cancer cell; conformation; conformational state; conformer; experiment; experimental research; experimental study; gene product; glutamyl-prolyl-tRNA synthetase; hnRNP; hnRNP L; human VEGF protein; inhibitor; inhibitor/antagonist; insight; lFN-Gamma; mRNA; mRNA Translation; macrophage; member; miRNA; multicatalytic endopeptidase complex; new therapeutic target; novel; nuclease; ontogeny; pathway; prevent; preventing; proline-tRNA; prolyl-tRNA; protein expression; public health relevance; research study; residence; response; social role; stem; tRNA; tRNA Synthetase; tRNA, proline-; tumor; tumor growth; vascular; vascular endothelial growth factor A, human; vascular endothelial growth factor, human; vertebrata
Project start date: 2009-01-01
Project end date: 2012-12-31
Budget start date: 1-JAN-2010
Budget end date: 31-DEC-2010
PFA/PA: PA-07-070
5R01GM086430-02 (2010): $282600
Structural Determinants Of Ceruloplasmin Inflammatory Activity
Paul L Fox, Professor Of Molecular Medicine
Cleveland Clinic Foundation 9500 Euclid Ave. Cleveland, Oh 44195
Grant 5P01HL076491-040002 from National Heart, Lung, And Blood Institute IRG: HLBP
Abstract: Inflammation and macromolecule oxidation are inter-related processes that are major contributors to atherogenesis. An important role of Cp in inflammation is suggested by its increased plasma level during the acute phase reaction and by its synthesis by activated macrophages in sites of inflammation. Clinical studies have shown that elevated plasma Cp is a significant risk factor for atherosclerosis, restenosis after endarterectomy, and myocardial infarction. In view of our finding that Cp copper induces oxidative modification of LDL in vitro, Cp may represent an important molecular link connecting inflammation, oxidation, and atherosclerosis. In new preliminary studies, we show that Cp binds with high affinity to LDL, and that the interaction is required for LDL oxidation by Cp. Other preliminary evidence indicates that Cp binds LDL in human plasma, and that binding to LDL may be higher in patients with cardiovascular disease. Studies with epitope-specific antibodies to Cp localized the interaction site to the second major sequence domain of Cp. These structural studies may be relevant to human inflammatory diseases, e.g., atherosclerosis, since coding-altering, single nucleotide polymorphisms (SNPs) have been identified in the human Cp gene, and two are adjacent to the Cp/LDL binding site. Preliminary studies in mice with targeted deletion of the Cp gene suggests that Cp increases oxidative modification of proteins and lipids in inflammatory sites in vivo. From previous work and our new preliminary results we propose the following hypothesis That in sites of inflammation, macrophage-derived Cp binds to LDL and causes specific copper ion-mediated oxidative modification of the protein and lipid components. We further propose that by defining the specific Cp domains and amino acids required for pro- and anti-inflammatory activities, recombinant, monofunctional Cp can be generated that can test the function of the individual activities in vivo. Finally, we propose that human Cp containing SNPs in these functional domains alter the inflammatory properties of Cp, and also the risk for atherosclerosis in humans. We will test this hypothesis by pursuing the following Specific Aims Aim 1. Determine the structural requirements for pro- and anti-inflammatory Cp activities; we will analyze structural determinants of Cp required for LDL oxidase, ferroxidase, and nitrosation activities, and for Cp interaction with LDL. Aim 2. Investigate the role of Cp in oxidation during inflammatory processes in vivo. Cp-null mice will be subjected to sepsis and peritonitis models of inflammation, and specific lipid and protein oxidation products in peritoneal lavage and plasma will be measured by LC-mass spectrometry These studies will elucidate, at the molecular, animal, and patient level, the role of Cp activities in inflammation.
Keywords: atherosclerosis, copper, enzyme activity, ferroxidase, inflammation, binding site, gene expression, low density lipoprotein, macromolecule, oxidation, peritonitis, plasma, recombinant protein, septicemia, single nucleotide polymorphism, SDS polyacrylamide gel electrophoresis, genetically modified animal, laboratory mouse, liquid chromatography mass spectrometry, polymerase chain reaction
Sponsored Links Excellgen http://Excellgen.com
Membrane Polarization And Endothelial Cell Motility
Paul L Fox, Professor Of Molecular Medicine
Cleveland Clinic Lerner Col/med-cwru P84 Cleveland, Oh 44195
Grant 5R01HL075255-05 from National Heart, Lung, And Blood Institute IRG: PC
Abstract: Endothelial cell (EC) movement is initiated by angiogenic growth factors, which trigger a sequence of spatially polarized intracellular events including the activation of motility-regulating small GTPases and the assembly of actin-dependent, force-generating systems at the cell anterior. Our primary interest is the role of the plasma membrane in cell movement. We have shown that membrane microviscosity is a key determinant of motility, and that basic fibroblast growth factor increases EC plasma membrane microviscosity as measured by fluorescence recovery after photobleaching (FRAP). Spatial analysis shows a highly polarized gradient of microviscosity in plasma membranes of rapidly migrating EC, with a leading edge that is substantially more viscous than the trailing edge. An important role of cholesterol in generation of this membrane microviscosity gradient is suggested by an increase in cholesterol content of the membrane, by gradient reversal upon cholesterol removal, and by relocalization of a fluorescent cholesterol analog, NBD-cholesterol, to the front of moving ECs. In studies of the mechanism that drives membrane polarization we have observed that caveolin-1, an intracellular cholesterol transport protein, is also highly polarized and accumulates in the rear of migrating ECs. In studies of the mechanism by which membrane physical properties regulate motility, we have found that increased membrane microviscosity increases the binding of Racl to plasma membranes in the front of moving ECs. We have also found that the ability of actin to deform large unitamellar vesicles is decreased when microviscosity is high, i.e., at an elevated cholesterol-to-phospholipid ratio. From these data we propose as a hypothesis that angiogenic growth factors alter cholesterol synthesis and trafficking to increase the membrane microviscosity at the leading edge of the moving cell. We further propose that increased microviscosity increases cell movement by increasing Racl-binding to the plasma membrane and by altering the barrier properties to improve the efficiency by which actin filaments propel the cell forward. We will test this hypothesis in three Specific Aims (1) Determine the molecular mechanism regulating polarization of membrane microviscosity during EC movement, (2) determine the mechanism by which microviscosity regulates Racl binding to membranes and (3) determine the role of membrane microviscosity in regulation of actin filament formation and function. The experiments will make use of cultured cells expressing GFP-tagged These studies will provide basic information on mechanisms regulating cell motility and may lead, in the long-term, to molecular strategies to inhibit or enhance cell migration. Pharmacological agents based on these results may be useful for inhibition of tumor angiogenesis or to enhance collateral blood vessel formation and the healing of synthetic vascular grafts.
Keywords: cell membrane, cell motility, membrane activity, vascular endothelium, P glycoprotein, actin, caveolin, cholesterol, growth factor, phosphorylation, steroid biosynthesis, viscosity, MCF7 cell, Sf9 cell line, affinity chromatography, atomic force microscopy, autoradiography, fluorescence microscopy, fluorescence recovery after photobleaching, green fluorescent protein, immunoprecipitation, polymerase chain reaction, western blotting
Project start date: 2004-01-01
Project end date: 2008-12-31
5R01HL075255-05 (2007): $362679
5R01HL075255-04 (2006): $373511
5R01HL075255-03 (2005): $382500
7R01HL075255-02 (2004): $382500
NOVEL MOUSE MODELS OF INFLAMMATION-RESOLUTION
Paul L Fox
Cleveland Clinic Lerner Col/med-cwru, Jjn5-01, Cleveland, Oh 44195
Grant 5RC1HL099353-02 from National Heart, Lung, And Blood Institute
Abstract: Chronic inflammation is a major contributor to the onset and progression of atherosclerosis and other vascular disorders. Studies of inflammation have focused primarily on the initiating processes and less attention has been given to the mechanisms by which inflammation is terminated. During the last several years a new paradigm has been invoked in which endogenous mediators limit or reduce these responses as part of an active "resolution of chronic inflammation" process. In our own studies we have described the discovery of a novel translational control system that has the features of an endogenous regulator of the inflammatory response in myeloid cells. We have shown that interferon (IFN)-gamma, a classic activator of monocyte/macrophages, induces assembly of the IFN-Gamma-Activated Inhibitor of Translation (GAIT) complex that binds a specific RNA element in the 3´untranslated region (UTR) of target mRNAs, e.g., vascular endothelial growth factor (VEGF)-A, and inhibits their translation. Remarkably, the GAIT complex consists of four abundant, "house-keeping" proteins especially recruited for this function Glu-Pro-tRNA synthetase (EPRS), ribosomal protein L13a, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and NS1-associated protein (NSAP1 or hnRNP Q). The complex assembles in two stages An early, pre-GAIT complex of unknown function that contains EPRS and NSAP1, and a late, functional GAIT complex that contains all four proteins. We have elucidated the critical phosphorylation sites essential for GAIT complex activation, namely, Ser77 in L13a and Ser999 in EPRS. Also, we have reported that interaction of NSAP1 acts as an early negative regulator of EPRS binding to target RNAs, and that the inhibition is overcome by late joining of L13a and GAPDH to form an active GAIT complex. In new Preliminary Studies we show that the GAIT pathway is functional in mouse macrophages; however, the mouse GAIT complex is heterotrimeric, lacking NSAP1 and early negative regula- tion. We propose to take advantage of this new information to generate a comprehensive ensemble of genetically-altered mice with mutations in key phosphorylation sites of GAIT components that inactivate, constitutively activate, or modulate, GAIT function in vivo. We will investigate gene expression and the role of the GAIT pathway in atherosclerosis using the well-established apoE-null mouse model. Generation of these mouse models will permit us and others to investigate the role of post-transcriptional gene regulation in the resolution of inflammation, and also the pathological consequences resulting from its dysregulation. These experiments will reveal insights into post-transcriptional mechanisms that regulate the protein expression pro- file of inflammatory macrophages, and the models will provide a unique resource for rigorous analysis of the causes of inflammation-resolution and the consequences of its dysregulation. Chronic inflammation is a major contributor to the onset and progression of atherosclerosis and other vascular disorders. Our laboratory has discovered a new pathway, the interferon-gamma-activated inhibitor of translation (GAIT) pathway, that functions to reduce or resolve inflammation by inhibiting the synthesis of pro-inflammatory proteins. We propose to develop genetically-modified mice to test the role of the GAIT pathway in preventing chronic inflammatory, cardiovascular disorders such as atherosclerosis. These models may reveal alternative targets for novel anti-inflammatory therapeutic strategies that, because of their specificity, may exhibit minimal adverse side-effects
Keywords: 3` Untranslated Regions; 3`UTR; 3-Phosphoglyceraldehyde; APOE [{C0003595}]; Abscission; Address; Adverse effects; Amino Acyl T RNA Synthetases; Amino Acyl-tRNA Ligases; Amino Acyl-tRNA Synthetases; Aminoacyl Transfer RNA Synthetase; Aminoacyl-tRNA Synthetase; Animal Model; Animal Models and Related Studies; Anti-Inflammatories; Anti-Inflammatory Agents; Anti-inflammatory; Antiinflammatories; Antiinflammatory Agents; Apo-E; ApoE; Apolipoprotein E; Area; Atheroscleroses; Atherosclerosis; Atherosclerotic Cardiovascular Disease; Attention; Autoregulation; Binding; Binding (Molecular Function); Binding Sites; Blood Vessels; Blood monocyte; Cardiovascular Diseases; Cells; Chronic; Combining Site; Complex; Cytokine Signal Transduction; Cytokine Signaling; D-Glyceraldehyde-3-phosphate[{..}]NADP+ oxidoreductase; Excision; Exhibits; Expression Profiling; Expression Signature; Extirpation; Family; GAPD; Gamma interferon; Gene Action Regulation; Gene Expression; Gene Expression Regulation; Gene Products, RNA; Gene Regulation; Gene Regulation Process; Generations; Genes; Genetic Alteration; Genetic Change; Genetic defect; Glyceraldehyde 3-Phosphate; Glyceraldehyde-3-Phosphate Dehydrogenases; Glyceraldehydephosphate Dehydrogenase; HBGF; Heterogeneous-Nuclear Ribonucleoproteins; Homeostasis; Housing; Human; Human, General; IFN; IFN-Gamma; IFN-g; IFNG; INFLM; In element; Indium; Inflammation; Inflammation Process; Inflammation Process, Multicellular; Inflammatory; Inflammatory Response; Informofer; Interferon Gamma; Interferon Type II; Interferon gamma (human lymphocyte protein moiety reduced); Interferon, Immune; Interferon-gamma; Interferons; Investigation; Knock-in; Knock-in Mouse; Knockout Mice; Laboratories; Lesion; Mammals, Mice; Man (Taxonomy); Man, Modern; Marrow monocyte; Mediator; Mediator of Activation; Mediator of activation protein; Messenger RNA; Mice; Mice, Knock-out; Mice, Knockout; Modeling; Molecular Fingerprinting; Molecular Interaction; Molecular Profiling; Murine; Mus; Mutate; Mutation; Myelogenous; Myeloid; Myeloid Cells; Nature; Null Mouse; Pathway interactions; Phosphoglyceraldehyde Dehydrogenase; Phosphorylation Site; Physiological Homeostasis; Predisposition; Predisposition gene; Pro-tRNA; Process; Propanal, 2-hydroxy-3-(phosphonooxy)-; Proteins; RNA; RNA, Messenger; RNA, Non-Polyadenylated; Reactive Site; Recruitment Activity; Regulation; Removal; Reporting; Research Resources; Resistance; Resolution; Resources; Ribonucleic Acid; Ribosomal Proteins; Role; Signal Pathway; Site; Specificity; Staging; Surgical Removal; Susceptibility; Susceptibility Gene; System; System, LOINC Axis 4; Technology; Testing; Therapeutic; Transfer RNA Synthetase; Transgenic Animals; Translations; Trauma; Treatment Side Effects; Triosephosphate Dehydrogenase; VEGFs; Vascular Diseases; Vascular Disorder; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vasculotropin; Vegf; ing; aminoacid tRNA ligase; atheromatosis; atherosclerotic vascular disease; blood vessel disorder; cardiovascular disorder; experiment; experimental research; experimental study; gene product; genome mutation; glyceraldehyde phosphate; hnRNP; in vivo; inhibitor; inhibitor/antagonist; insight; lFN-Gamma; mRNA; macrophage; model organism; molecuar profile; molecular signature; monocyte; mouse model; novel; pathway; predisposing gene; prevent; preventing; proline-tRNA; prolyl-tRNA; protein expression; recruit; research study; resection; resistant; response; side effect; social role; tRNA Synthetase; tRNA, proline-; therapy adverse effect; treatment adverse effect; vascular
Relevance: Relevance Chronic inflammation is a major contributor to the onset and progression of atherosclerosis and other vascular disorders. Our laboratory has discovered a new pathway, the interferon-gamma-activated inhibitor of translation (GAIT) pathway, that functions to reduce or resolve inflammation by inhibiting the synthesis of pro- inflammatory proteins. We propose to develop genetically-modified mice to test the role of the GAIT pathway in preventing chronic inflammatory, cardiovascular disorders such as atherosclerosis. These models may reveal alternative targets for novel anti-inflammatory therapeutic strategies that, because of their specificity, may exhibit minimal adverse side-effects
Project start date: 2009-09-30
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: RFA-OD-09-003
5RC1HL099353-02 (2010): $332091
1RC1HL099353-01 (2009): $457917
GLOBAL ANALYSIS OF MRNA POLARIZATION IN MIGRATING ENDOTHELIAL CELLS
Paul L Fox, Professor
Cleveland Clinic Lerner Col/med-cwru, Jjn5-01, Cleveland, Oh 44195
Grant 5R21HL094841-02 from National Heart, Lung, And Blood Institute
Abstract: Blood vessels are exposed to multiple stresses that can induce injury to the endothelium, which must undergo rapid repair to restore normal function of the vessel and the entire cardiovascular system. Endothelial cell (EC) migration has a critical role in this repair process, as well as in formation of new blood vessels. Polarization of multiple cell systems, including signaling, adhesion proteins, plasma membrane, secretion processes, cyto- skeletal arrangement, etc., is crucial for induction and control of cell migration. Recent studies have shown that 2-actin mRNA, and several other motility-related transcripts, accumulated in the front of moving cells, but little is known about the total ensemble of polarized mRNAs and their common localization elements. Our long-term goal is to understand the contribution of mRNA polarization to the development of asymmetry during EC movement, and the mechanisms by which mRNAs are polarized in planar moving cells. To accomplish this goal, we have initiated a comprehensive, global analysis of the mRNAs that become polarized during EC migration. We have applied the laser microdissection methodology to capture cell fragments from forward and rearward regions of planar-moving EC, a model representing the wound-healing process. We propose to couple this microdissection method with microarray technologies, followed by rigorous biochemical investigation to address the following hypothesis cis-elements in mRNAs determine their localization in both the front and back of planar-migrating EC, and are responsible for targeting proteins to their site of function. We will test this hypothesis by pursuing the following specific aims (1) identify novel mRNAs polarized in migrating EC, and (2) investigate RNA elements responsible for mRNA polarization during cell migration. Our studies will provide the first global analysis of mRNA localization during cell migration, and it will begin to fill important gaps in our understanding of cell polarization during migration. In particular, little is known about the RNA elements that induce mRNAs to accumulate in the cell front and virtually nothing is known about mRNAs (and their functional elements) that accumulate in the cell rear. In addition to the mechanistic contribution to our understanding of cell polarization, our work can reveal new insights into the regulation of cell movement, and has the potential of new interventions to alter motility. For example, identification of novel localization elements presents novel therapeutic targets for modulating mRNA localization and cell migration by RNA-based approaches, for example, by treatment with antisense RNA oligomers targeting the specific RNA element. Finally, our work is likely to provide and validate a technological advance that will permit global analysis of asymmetric mRNA in other systems. Blood vessels are exposed to multiple stresses that can injure the endothelial cell (EC) lining of the vessel wall. Subsequent migration of EC is critical for vessel wall repair and restoration of normal blood vessel function. Polarization of the messenger RNAs (mRNA) encoding movement-related proteins is a newly discovered event that may contribute to cell directionality required for EC movement. Our studies will provide the first global analysis of mRNA polarization during planar cell migration, and it may reveal novel therapeutic targets for modulating localization of motility-related mRNAs and cell migration
Keywords: 2-dimensional; Abbreviations; Actins; Address; Adhesions; Aldehydes; Anti-Sense RNA; Antisense RNA; Back; Band Shift Mobility Assay; Bandshift Mobility Assay; Binding; Binding (Molecular Function); Binding Proteins; Bio-Informatics; Biochemical; Bioinformatics; Blood Vessels; Bovine Species; Cardiovascular; Cardiovascular Body System; Cardiovascular system; Cardiovascular system (all sites); Cattle; Cell Communication and Signaling; Cell Line; Cell Lines, Strains; Cell Locomotion; Cell Migration; Cell Movement; Cell Signaling; Cell membrane; Cell surface; CellLine; Cells; Cellular Migration; Chimera; Chimera organism; Collecting Cell; Confocal Microscopy; Cytoplasmic Membrane; Data; Data Banks; Data Bases; Databank, Electronic; Databanks; Database, Electronic; Databases; Dehydrogenases; Detection; Development; Dorsum; Electromagnetic, Laser; Electrophoretic Mobility Shift Assay; Elements; Endothelial Cells; Endothelium; Energy Expenditure; Energy Metabolism; Event; FISH Technic; FISH Technique; FISH analysis; Family; Fluorescence Photobleaching Recovery; Fluorescence Polarization; Fluorescence Recovery After Photobleaching; Fluorescent in Situ Hybridization; GFP; Gait; Gamma interferon; Gene Expression; Gene Products, RNA; Generations; Genome; Goals; Green Fluorescent Proteins; Healed; IFN; IFN-Gamma; IFN-g; IFNG; In Situ Hybridization, Fluorescence; In element; Indium; Individual; Injury; Inositide Phospholipids; Inositol Phosphoglycerides; Inositol Phospholipids; Interferon Gamma; Interferon Type II; Interferon gamma (human lymphocyte protein moiety reduced); Interferon, Immune; Interferon-gamma; Interferons; Intervention; Intervention Strategies; Intracellular Communication and Signaling; Investigation; Label; Lasers; Lead; Ligand Binding Protein; Medical Imaging, Positron Emission Tomography; Messenger RNA; Method LOINC Axis 6; Methodology; Methods; Microarray Analysis; Microarray-Based Analysis; Microdissection; Mobility Shift Assay; Modeling; Molecular Interaction; Motility; Motility, Cellular; Movement; Organ System, Cardiovascular; Oxidoreductase; PET; PET Scan; PET imaging; PETSCAN; PETT; Pb element; Phosphates; Phosphatidyl Inositol; Phosphatidylinositols; Phosphoinositides; Plasma Membrane; Polyethylene Terephthalates; Positron Emission Tomography Scan; Positron-Emission Tomography; Process; Proteins; Proton Magnetic Resonance Spectroscopic Imaging; PtdIns; RNA; RNA, Messenger; RNA, Non-Polyadenylated; RT-PCR; RTPCR; Rad.-PET; Radiation, Laser; Reductases; Regulation; Reporter; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic Acid; Role; Sampling; Signal Transduction; Signal Transduction Systems; Signaling; Site; Stress; Structure; System; System, LOINC Axis 4; Testing; Therapeutic; Transcript; Transfection; Translations; UTRs; Untranslated Regions; VEGFs; Vascular Endothelial Growth Factors; Vascular, Heart; Vegf; Work; Wound Healing; Wound Repair; actin 2; actin2; base; biological signal transduction; body movement; bovid; bovine; cell motility; circulatory system; clinical data repository; clinical data warehouse; cow; cultured cell line; data repository; deletion analysis; experiment; experimental research; experimental study; gel shift assay; gene product; healing; heavy metal Pb; heavy metal lead; inhibitor; inhibitor/antagonist; injured; inorganic phosphate; insight; interventional strategy; lFN-Gamma; mRNA; microarray technology; migration; new therapeutic target; novel; plasmalemma; protein complex; protein protein interaction; public health relevance; relational database; repair; repaired; research study; restoration; reverse transcriptase PCR; secretion process; skeletal; social role; tissue repair; two-dimensional; vascular
Relevance: Relevance Blood vessels are exposed to multiple stresses that can injure the endothelial cell (EC) lining of the vessel wall. Subsequent migration of EC is critical for vessel wall repair and restoration of normal blood vessel function. Polarization of the messenger RNAs (mRNA) encoding movement-related proteins is a newly discovered event that may contribute to cell directionality required for EC movement. Our studies will provide the first global analysis of mRNA polarization during planar cell migration, and it may reveal novel therapeutic targets for modulating localization of motility-related mRNAs and cell migration
Project start date: 2009-07-20
Project end date: 2011-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-06-181
5R21HL094841-02 (2010): $196250
1R21HL094841-01A1 (2009): $235500
FGF RECEPTOR SIGNALING IN BREAST CANCER ANGIOGENESIS
Paul L Fox, Professor Of Molecular Medicine
Cleveland Clinic Foundation 9500 Euclid Ave. Cleveland, Oh 44195
Grant 5R01HL054519-04 from National Heart, Lung, And Blood Institute IRG: ZHL1
Abstract: Angiogenesis, or the formation of new blood vessels, is believed to be required for growth of tumors of the breast beyond a maximum size of 1-2 mm. The factors that regulate tumor angiogenesis are incompletely understood but directed endothelial cell (EC) movement, followed by proliferation of trailing EC, appears to initiate the formation of capillaries during normal vessel development and tumor angiogenesis. Tumor-derived basic fibroblast growth factor (bFGF) may be an important initiator of tumor angiogenesis since the factor potently stimulates EC movement in vitro and since a significant fraction of breast cancer tumors exhibit amplification of FGF- or FGF receptor-related genes and expression of their protein products. The signal transduction pathway(s) responsible for bFGF-stimulated movement is not known; however, we have evidence that the activity of a novel, 56 kDa pertussis toxin-sensitive G-protein is required. This G-protein (designated G/m, or putative motility-related G- protein) is physically associated with the flg-type FGF receptor, FGFR-1 and is activated by bFGF to bind GTP. G/m is required for maximal activation of the cytosolic form of phospholipase A/2 (cPLA/2), and the arachidonate released (or a downstream metabolite) is necessary for bFGF- mediated EC movement. The long-term goal of our research is to understand this pathway in detail, and in particular, to elucidate the role of Gm in angiogenic responses of EC. We propose as a hypothesis that breast tumor cells secrete bFGF which initiates migratory processes in EC of nearby blood vessels. We also propose that the interaction of bFGF with FGFR-1 activates G/m by increasing GTP-binding, and that G/m-GTP activates cPLA/2 to release arachidonate, the precursor of a promigratory eicosanoid(s). We will test this hypothesis by pursuing the following Specific Aims (1) map the site on FGFR-1 that interacts with G/m, (2) purify G/m from fetal bovine brain, (3) characterize the functional and kinetic properties of purified G/m, and (4) investigate the presence and location of G/m in breast cancer tissues. Successful completion of these Specific Aims will provide important molecular information on the signaling pathways regulating bFGF-stimulated EC motility. The elucidation of the intracellular pathways that regulate EC movement, and therefore the initial steps of tumor angiogenesis, may provide a foundation for the development of therapies that interfere with these processes in vivo. Since the signaling events initiated by diverse growth factors may converge to a single critical pathway regulating cell movement, our approach may have particular significant in view of the ability of transformed cells able to secrete multiple growth- and motility-inducing agents. Identification of G/m as a factor involved in early breast cancer may also have diagnostic value in guiding the course of surgical adjuvant therapy to optimize patient outcome.
Keywords: angiogenesis, biological signal transduction, breast neoplasm, fibroblast growth factor, growth factor receptor, G protein, genetic mapping, guanine nucleotide binding protein, molecular pathology, protein purification, receptor expression, recombinant protein, vascular endothelium, human tissue, tissue /cell culture, transfection, western blotting
Project start date: 1995-06-01
Project end date: 2000-05-31
5R01HL054519-04 (1998): $214740
5R01HL054519-03 (1997): $206481
Sponsored Links Excellgen http://Excellgen.com
ROLE OF CERULOPLASMIN IN IRON METABOLISM AND OVERLOAD
Paul L Fox, Professor Of Molecular Medicine
Cleveland Clinic Foundation
9500 Euclid Ave.
cleveland, Oh 44195
Grant 5R01HL067725-02 from National Heart, Lung, And Blood Institute IRG: ZDK1
Abstract: adapted ) The importance of iron is underscored by its participation in many cellular processes involving oxygen or redox reactions. Iron in excess of cellular needs is toxic; dietary overload or hereditary hemochromatosis leads to tissue iron deposition and injury, most likely due to redox activity of iron and consequent free radical reactions. The precise balance required to maintain appropriate cellular and tissue iron levels has led to mechanisms that regulate the synthesis of iron transport and storage proteins, e.g., transferrin receptor and ferritin. A role for copper in iron metabolism has been known for about 70 years. The important role for ceruloplasmin (Cp) in iron metabolism in vivo has been reinforced by the identification of "aceruloplasminemia" patients with Cp gene defects and massive iron deposits in many tissues, including the brain. This function for Cp has received support from studies showing that two Cp homologues, fet3p in yeast and hephaestin in mouse, play key roles in iron homeostasis. We have shown that the rate of Cp synthesis by HepG2 and Hep3B cells is tightly regulated by cellular iron status. Iron deficiency markedly increases Cp protein synthesis and gene expression. Nuclear "run-on" and mRNA stability studies indicate that regulation is by a transcriptional mechanism. We have new evidence that Cp transcription is regulated by hypoxia-inducible factor (HIF)-1 responsive elements since transcription is regulated by hypoxia and other HIF-1 activators. In addition, an enhancer element in the human Cp gene 5´-regulatory region contains HIF-1 responsive elements which increase reporter gene expression about 10-fold. In contrast to the stimulatory activity of iron deficiency, excess iron decreases Cp synthesis of HepG2 cells; surprisingly, a post-transcriptional mechanism has been observed in which Cp mRNA is destabilized by iron. These results support the important role of Cp in maintenance of cellular iron homeostasis. We hypothesize that the rate of hepatic Cp synthesis, like that of other proteins involved in iron transport, is regulated by cell iron status. In this application we will test the following specific aims that a specific trans-activating factor(s) in iron-deficient HepG2 cells binds to a cis-acting element in the Cp 5´-regulatory region, thereby increasing Cp transcription. Furthermore, iron in excess alters the activity of a specific trans-acting factor(s) that binds to a region of the Cp 3´-UTR, thereby destabilizing the Cp mRNA. The long-term goal of this research is to understand the specific function of Cp in iron homeostasis, and especially its role in primary (genetic) and secondary (dietary) iron overload states
Keywords: ferroxidase, iron metabolism, iron poisoning genetic regulatory element, genetic transcription, hypoxia, liver metabolism, messenger RNA, posttranscriptional RNA processing, transcription factor nuclear runoff assay, tissue /cell culture
Project start date: 2000-09-27
Project end date: 2004-07-31
5R01HL067725-02 (2001): $259000
1R01HL067725-01 (2000): $259000
3R01HL067725-03S1 (2003): $36982
7R01HL067725-04 (2003): $332963
Paul L Fox
Cleveland Clinic Lerner Col/med-cwru
Project start date: 2009-01-01
Project end date: 2012-12-31
OXIDANT ROLE OF CERULOPLASMIN IN ATHEROSCLEROSIS
Paul L Fox, Professor Of Molecular Medicine
Cleveland Clinic Foundation 9500 Euclid Ave. Cleveland, Oh 44195
Grant 5R01HL052692-02 from National Heart, Lung, And Blood Institute IRG: HEM
Project start date: 1994-12-01
Project end date: 1997-11-30
5R01HL052692-02 (1996): $197364
FGF RECEPTOR SIGNALING IN BREAST CANCER ANGIOGENESIS
Paul L Fox, Professor Of Molecular Medicine
Cleveland Clinic Foundation 9500 Euclid Ave. Cleveland, Oh 44195
Grant 5R01HL054519-02 from National Heart, Lung, And Blood Institute IRG: ZHL1
Project start date: 1995-06-01
Project end date: 1999-05-31
5R01HL054519-02 (1996): $198537