RESUMEN
To study the possible role of the human lipid-oxidizing enzyme 15-lipoxygenase (15-LO) in atherosclerosis, we overexpressed it specifically in the vascular wall of C57B6/SJL mice by using the murine preproendothelin-1 promoter. The mice overexpressing 15-LO were crossbred with low density lipoprotein (LDL) receptor-deficient mice to investigate atherogenesis. High levels of 15-LO were expressed in the atherosclerotic lesion in the double-transgenic mice as assessed by immunohistochemistry. The double-transgenic, 15-LO-overexpressing, LDL receptor-deficient mice (LDLR-/-/15LO) developed significantly larger atherosclerotic lesions at the aortic sinus compared with lesions in the LDL receptor-deficient (LDLR-/-) mice after 3 and 6 weeks (107,000 versus 28,000 microm(2) [P:<0.001] and 121,000 versus 87,000 microm(2) [P:<0.05], respectively) of an atherogenic diet. LDL from the LDLR-/-/15LO mice was more susceptible to oxidation than was the LDL from the control LDLR-/- mice, as shown by a shorter lag period for copper-induced conjugated diene formation. On the other hand, no differences were found in the levels of serum anti-oxidized LDL antibodies between the study groups. There were also no differences with respect to the density of macrophages and T lymphocytes infiltrating the lesions in both experimental groups. Taken together, these results support the hypothesis that 15-LO overexpression in the vessel wall is associated with enhanced atherogenesis.
Asunto(s)
Araquidonato 15-Lipooxigenasa/fisiología , Arteriosclerosis/enzimología , Endotelio Vascular/enzimología , Receptores de LDL/metabolismo , Animales , Anticuerpos/sangre , Araquidonato 15-Lipooxigenasa/biosíntesis , Endotelina-1 , Endotelinas/genética , Lípidos/sangre , Lipoproteínas/metabolismo , Lipoproteínas LDL/inmunología , Ratones , Ratones Transgénicos , Oxidación-Reducción , Precursores de Proteínas/genética , Receptores de LDL/deficienciaRESUMEN
Despite numerous studies, the precise role of dietary n-6 polyunsaturated fatty acids in the pathogenesis of atherosclerosis remains controversial. It has been shown that feeding an n-6-enriched diet resulted in decreased atherosclerosis in African green monkeys and was associated with a reduction in LDL levels. However, other authors reported that n-6 supplementation increased the oxidative stress and the susceptibility of LDL to undergo in vitro oxidation, thus potentially enhancing atherosclerosis. The present study was designed to investigate the effect of dietary supplementation of n-6 polyunsaturated fats (safflower oil), as compared with a saturated fat-rich diet (Paigen), on the blood lipid profile and atherosclerosis in two mouse models. In the first experiment, female C57BL/6 mice (n=23-30 per group) were fed a cholate containing Paigen diet, a safflower oil-rich diet (with cholate), or normal chow for 15 weeks. No significant differences between the high fat diet groups were evident with respect to total cholesterol, LDL, HDL or triglyceride levels. The extent of aortic sinus fatty streaks did not differ significantly between the two groups. In the second experiment, LDL-receptor-deficient (LDL-RD) mice (n=20-30 per group) were randomized into similar dietary regimens. Mice consuming a safflower oil-enriched diet developed significantly less atherosclerosis, in comparison with Paigen diet-fed mice. A reduction in LDL levels, although not of a similar magnitude as the reduction in atherosclerosis, was evident in the safflower oil-fed mice when compared to the Paigen diet-fed littermates. In both mouse models of atherosclerosis, LDL isolated from the plasma of mice on the n-6 polyunsaturated diet was rendered slightly more susceptible to oxidation in vitro, as indicated by a shorter lag period for diene formation. Thus, the effects of n-6 fatty acids on the lipoprotein composition and other potential influences may have contributed to the anti-atherogenic effect in the LDL-RD mouse model.
Asunto(s)
Arteriosclerosis/dietoterapia , Grasas Insaturadas en la Dieta/administración & dosificación , Suplementos Dietéticos , Ácidos Grasos Insaturados/administración & dosificación , Lipoproteínas LDL/sangre , Estrés Oxidativo/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , Aorta/patología , Arteriosclerosis/sangre , Arteriosclerosis/inducido químicamente , Arteriosclerosis/patología , Peso Corporal , Dieta Aterogénica , Progresión de la Enfermedad , Ácidos Grasos Omega-6 , Femenino , Lipoproteínas LDL/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Receptores de LDL/sangre , Receptores de LDL/deficiencia , Receptores de LDL/efectos de los fármacos , Aceite de Cártamo/administración & dosificación , Triglicéridos/sangreRESUMEN
Monocyte chemoattracant-1 (MCP-1) stimulates leukocyte chemotaxis to inflammatory sites, such as rheumatoid arthritis, atherosclerosis, and asthma, by use of the MCP-1 receptor, CCR2, a member of the G-protein-coupled seven-transmembrane receptor superfamily. These studies identified a family of antagonists, spiropiperidines. One of the more potent compounds blocks MCP-1 binding to CCR2 with a K(d) of 60 nm, but it is unable to block binding to CXCR1, CCR1, or CCR3. These compounds were effective inhibitors of chemotaxis toward MCP-1 but were very poor inhibitors of CCR1-mediated chemotaxis. The compounds are effective blockers of MCP-1-driven inhibition of adenylate cyclase and MCP-1- and MCP-3-driven cytosolic calcium influx; the compounds are not agonists for these pathways. We showed that glutamate 291 (Glu(291)) of CCR2 is a critical residue for high affinity binding and that this residue contributes little to MCP-1 binding to CCR2. The basic nitrogen present in the spiropiperidine compounds may be the interaction partner for Glu(291), because the basicity of this nitrogen was essential for affinity; furthermore, a different class of antagonists, a class that does not have a basic nitrogen (2-carboxypyrroles), were not affected by mutations of Glu(291). In addition to the CCR2 receptor, spiropiperidine compounds have affinity for several biogenic amine receptors. Receptor models indicate that the acidic residue, Glu(291), from transmembrane-7 of CCR2 is in a position similar to the acidic residue contributed from transmembrane-3 of biogenic amine receptors, which may account for the shared affinity of spiropiperidines for these two receptor classes. The models suggest that the acid-base pair, Glu(291) to piperidine nitrogen, anchors the spiropiperidine compound within the transmembrane ovoid bundle. This binding site may overlap with the space required by MCP-1 during binding and signaling; thus the small molecule ligands act as antagonists. An acidic residue in transmembrane region 7 is found in most chemokine receptors and is rare in other serpentine receptors. The model of the binding site may suggest ways to make new small molecule chemokine receptor antagonists, and it may rationalize the design of more potent and selective antagonists.
Asunto(s)
Citocinas , Receptores de Citocinas/antagonistas & inhibidores , Receptores de Citocinas/química , Inhibidores de Adenilato Ciclasa , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Calcio/metabolismo , Línea Celular , Quimiocina CCL5/antagonistas & inhibidores , Quimiocina CCL7 , Quimiotaxis , Cricetinae , AMP Cíclico/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Glutámico/química , Concentración 50 Inhibidora , Cinética , Ligandos , Luciferasas/metabolismo , Datos de Secuencia Molecular , Proteínas Quimioatrayentes de Monocitos/antagonistas & inhibidores , Mutagénesis Sitio-Dirigida , Nitrógeno/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Receptores CCR2 , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Citocinas/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Transfección , Células Tumorales CultivadasRESUMEN
Abundant data is present to implicate oxidatively modified low-density lipoprotein (oxLDL) in enhanced atherogenesis. Among the factors involved in LDL oxidation, an important role has been attributed to human 15-lipoxygenase (LO) and its murine analog 12-LO. The expression of these peroxidizing enzymes is under the control of cytokines, the principal of which is IL-4. In the present study we tested the hypothesis that knocking out the IL-4 gene from C57BL/6 mice would result in suppression of fatty streaks. For this purpose, we have fed 45 female IL-4 transgenic knockout (IL-4T KO) and 45 wild-type (WT) mice an atherogenic diet for 15 weeks. Consecutive determinations of the lipid profile from both study groups were performed at monthly intervals, and fatty streak formation was assessed at the aortic sinus level, upon sacrifice. The two study groups did not differ significantly with respect to the lipid profile or the uptake and degradation of iodinated oxLDL by their peritoneal macrophages. We found that the endogenous deficiency of IL-4 did not confer protection from early atherosclerosis in the IL-4T KO as compared to their WT littermates (determined at the aortic sinus). Immunohistochemical studies, Western blots and 12/15-LO activity assays revealed the presence and activity of 12/15-LO in macrophages of WT mice as well as in IL-4T KO mice. Both did not differ significantly between the study groups. The data from this study imply that deficiency in IL-4 does not affect early atherosclerosis in C57BL/6 mice fed a high-cholesterol diet.
Asunto(s)
Arteriosclerosis/etiología , Interleucina-4/deficiencia , Animales , Arteriosclerosis/metabolismo , Humanos , Interleucina-4/genética , Peroxidación de Lípido , Lipoproteínas LDL/metabolismo , Ratones , Ratones NoqueadosRESUMEN
The CC chemokine, monocyte chemotactic protein, 1 (MCP-1) functions as a major chemoattractant for T-cells and monocytes by interacting with the seven-transmembrane G protein-coupled receptor CCR2. To identify which residues of MCP-1 contribute to signaling though CCR2, we mutated all the surface-exposed residues to alanine and other amino acids and made some selective large changes at the amino terminus. We then characterized the impact of these mutations on three postreceptor pathways involving inhibition of cAMP synthesis, stimulation of cytosolic calcium influx, and chemotaxis. The results highlight several important features of the signaling process and the correlation between binding and signaling: The amino terminus of MCP-1 is essential as truncation of residues 2-8 ([1+9-76]hMCP-1) results in a protein that cannot stimulate chemotaxis. However, the exact peptide sequence may be unimportant as individual alanine mutations or simultaneous replacement of residues 3-6 with alanine had little effect. Y13 is also important and must be a large nonpolar residue for chemotaxis to occur. Interestingly, both Y13 and [1+9-76]hMCP-1 are high-affinity binders and thus affinity of these mutants is not correlated with ability to promote chemotaxis. For the other surface residues there is a strong correlation between binding affinity and agonist potency in all three signaling pathways. Perhaps the most interesting observation is that although Y13A and [1+9-76]hMCP are antagonists of chemotaxis, they are agonists of pathways involving inhibition of cAMP synthesis and, in the case of Y13A, calcium influx. These results demonstrate that these two well-known signaling events are not sufficient to drive chemotaxis. Furthermore, it suggests that specific molecular features of MCP-1 induce different conformations in CCR2 that are coupled to separate postreceptor pathways. Therefore, by judicious design of antagonists, it should be possible to trap CCR2 in conformational states that are unable to stimulate all of the pathways required for chemotaxis.
Asunto(s)
Aminoácidos/fisiología , Quimiocina CCL2/fisiología , Receptores de Quimiocina/fisiología , Receptores de Citocinas/fisiología , Transducción de Señal , Aminoácidos/aislamiento & purificación , Sitios de Unión/genética , Calcio/antagonistas & inhibidores , Calcio/metabolismo , Línea Celular , Membrana Celular/genética , Membrana Celular/fisiología , Inhibición de Migración Celular , Quimiocina CCL2/agonistas , Quimiocina CCL2/genética , AMP Cíclico/antagonistas & inhibidores , Humanos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Receptores CCR2 , Receptores de Quimiocina/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal/genética , Tirosina/genética , Tirosina/fisiologíaRESUMEN
The CC chemokine, MCP-1, has been identified as a major chemoattractant for T cells and monocytes, and plays a significant role in the pathology of inflammatory diseases. To identify the regions of MCP-1 that contact its receptor, CCR2, we substituted all surface-exposed residues with alanine. Some residues were also mutated to other amino acids to identify the importance of charge, hydrophobicity, or aromaticity at specific positions. The binding affinity of each mutant for CCR2 was assayed with THP-1 and CCR2-transfected CHL cells. The majority of point mutations had no effect. Residues at the N-terminus of the protein, known to be crucial for signaling, contribute less than a factor of 10 to the binding affinity. However, two clusters of primarily basic residues (R24, K35, K38, K49, and Y13), separated by a 35 A hydrophobic groove, reduced the level of binding by 15-100-fold. A peptide fragment encompassing residues 13-35 recapitulated some of the mutational data derived from the intact protein. It exhibited modest binding as a linear peptide and dramatically improved affinity when the region which adopts a single turn of a 3(10)-helix in the protein, which includes R24, was constrained by a disulfide bond. Additional constraints at the ends of the peptide, corresponding to the disulfide between the first and third cysteines in MCP-1, yielded further improvements in affinity. Together, these data suggest a model in which a large surface area of MCP-1 contacts the receptor, and the accumulation of a number of weak interactions results in the 35 pM affinity observed for the wild-type (WT) protein. The receptor binding site of MCP-1 also is significantly different from the binding sites of RANTES and IL-8, providing insight into the issue of receptor specificity. It was previously shown that the N-terminus of CCR2 is critical for binding MCP-1 [Monteclaro, F. S., and Charo, I. F. (1996) J. Biol. Chem. 271, 19084-92; Monteclaro, F. S., and Charo, I. F. (1997) J. Biol. Chem. 272, 23186-90]. Point mutations of six acidic residues in this region of the receptor were made to test their role in ligand binding. This identified D25 and D27 of the DYDY motif as being important. On the basis of our data, we propose a model in which the receptor N-terminus lies along the hydrophobic groove in an extended fashion, placing the DYDY motif near the basic cluster involving R24 and K49 of MCP-1. This in turn orients the signaling residues (Y13 and the N-terminus) for productive interaction with the receptor.
Asunto(s)
Quimiocina CCL2/química , Quimiocina CCL2/metabolismo , Receptores de Quimiocina/metabolismo , Receptores de Citocinas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Quimiocina CCL2/genética , Cricetinae , Cricetulus , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptores CCR2 , Receptores de Quimiocina/química , Receptores de Citocinas/químicaRESUMEN
To address the role of dimerization in the function of the monocyte chemoattractant protein-1, MCP-1, we mutated residues that comprise the core of the dimerization interface and characterized the ability of these mutants to dimerize and to bind and activate the MCP-1 receptor, CCR2b. One mutant, P8A*, does not dimerize. However, it has wild type binding affinity, stimulates chemotaxis, inhibits adenylate cyclase, and stimulates calcium influx with wild type potency and efficacy. These data suggest that MCP-1 binds and activates its receptor as a monomer. In contrast, Y13A*, another monomeric mutant, has a 100-fold weaker binding affinity, is a much less potent inhibitor of adenylate cyclase and stimulator of calcium influx, and is unable to stimulate chemotaxis. Thus Tyr13 may make important contacts with the receptor that are required for high affinity binding and signal transduction. We also explored whether a mutant, [1+9-76]MCP-1 (MCP-1 lacking residues 2-8), antagonizes wild type MCP-1 by competitive inhibition, or by a dominant negative mechanism wherein heterodimers of MCP-1 and [1+9-76]MCP-1 bind to the receptor but are signaling incompetent. Consistent with the finding that MCP-1 can bind and activate the receptor as a monomer, we demonstrate that binding of MCP-1 in the presence of [1+9-76]MCP-1 over a range of concentrations of both ligands fits well to a simple model in which monomeric [1+9-76]MCP-1 functions as a competitive inhibitor of monomeric MCP-1. These results are crucial for elucidating the molecular details of receptor binding and activation, for interpreting mutagenesis data, for understanding how antagonistic chemokine variants function, and for the design of receptor antagonists.
Asunto(s)
Quimiocina CCL2/metabolismo , Receptores de Quimiocina , Receptores de Citocinas/metabolismo , Dimerización , Disulfuros/química , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , Receptores CCR2 , Receptores de Citocinas/antagonistas & inhibidores , Proteínas Recombinantes/metabolismoRESUMEN
We have isolated a murine macrophage cDNA encoding a 12-lipoxygenase, that represents the homolog of the human 15-lipoxygenase. The predicted amino acid sequence of this lipoxygenase is highly similar to the rat 12-lipoxygenase isolated from brain and human 15-lipoxgenase. The recombinant enzyme expressed in Cos-7 cells oxidizes arachidonic acid to 12- and 15-HETE with a profile similar to that obtained from peritoneal macrophages. A polyclonal antibody generated against a putative peptide recognizes a 75 kDa protein in cell extracts from mouse peritoneal macrophages and transfected Cos-7 cells. The lipoxygenase cDNA hybridizes to a 2.5 kb mRNA present in peritoneal macrophages, lung, spleen, heart and liver. RT-PCR analysis indicates that the same lipoxygenase is expressed in mouse reticulocytes. A partial genomic clone for this lipoxygenase has also been characterized. Southern blot analysis of mouse genomic DNA indicates that this is a single copy gene.
Asunto(s)
Lipooxigenasa/genética , Macrófagos/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Clonación Molecular , ADN Complementario , Humanos , Lipooxigenasa/química , Ratones , Datos de Secuencia Molecular , Ratas , Alineación de SecuenciaRESUMEN
It is well accepted that high levels of low-density lipoprotein (LDL) cholesterol in the plasma are associated with increased risk of atherosclerosis. The cellular and molecular mechanisms linking the two however, have not been fully resolved. One of the processes involved in atherogensis that has been intensively studied in this regard is the oxidation of LDL. Oxidation may convert LDL into an atherogenic form, which incites an inflammatory and proliferative response characteristic of the atherosclerotic lesion. One of the potential mediators in this process is the lipid peroxidating enzyme 15-lipoxygenase, which has been shown to be induced in the atherosclerotic lesion and is capable of oxidizing LDL. In this article, we review the motivation for looking at mechanisms of LDL oxidation and the proposed involvement of 15-lipoxygenase in the pathogenesis of the disease.
RESUMEN
Mounting evidence suggests that oxidative processes contribute to the pathogenesis of atherosclerosis and that antioxidants may represent a strategy to complement the lowering of lipids in the therapy of this disease. Although multiple molecular events have been identified in vitro and although it is tempting to ascribe multiple atherogenic properties to oxidized LDL, our understanding of this process remains incomplete. Further research is warranted in several areas. First, it will be important to selectively inhibit different aspects of the process to determine the relative contribution of various biological targets. In this regard pharmacological inhibition of 15-lipoxygenase in vivo in relevant animal models is required to address the question of the contribution of this enzyme to significant oxidative events. The lack of specific inhibitors has made this task more difficult. It will also be important to define the biologically active moiety of oxidized LDL to begin to determine the mechanisms through which it exerts its atherogenic effects. It is likely that alternate protein targets can be identified both downstream and upstream of the oxidative process. Research is only now beginning to elucidate the inflammatory mechanisms that account for the cellular response. Further research into adhesion events, cytokine profiles, and downstream effector molecules of the oxidative process are likely to identify alternate targets for therapeutic intervention.
Asunto(s)
Arteriosclerosis/fisiopatología , Lipooxigenasa/metabolismo , Animales , Antioxidantes/farmacología , Araquidonato 15-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Arteriosclerosis/patología , Arteriosclerosis/prevención & control , Plaquetas/fisiología , Endotelio Vascular/fisiopatología , Humanos , Lipoproteínas LDL/sangre , Modelos Biológicos , Monocitos/fisiología , Oxidación-ReducciónRESUMEN
Five protein families are needed to encompass the diversity of cyclic-AMP (cAMP) phosphodiesterases (PDE). Family IV PDEs (PDE IV) specifically hydrolyze cAMP with a low Km, and are selectively inhibited by rolipram (Rp) and related drugs. Cloned cDNAs from rat (r) suggest that the PDE IV family comprises four distinct members, designated A, B, C and D. Using RN from a human lymphocytic B-cell line (43D-Cl2), we have isolated a 3.8-kb cDNA by low-stringency screening using a rat PDE IV member B (r-PDE IVB) probe. Expression of the human (h) cDNA in Escherichia coli results in cAMP-specific PDE activity that is Rp sensitive. A single large open reading frame (ORF) predicts a 564-amino-acid protein with 92.9% identity to r-PDE IVB; at the nucleotide level the identity is 86.3%. This h-PDE IVB clone, HPB106, differs from a related cDNA clone isolated by others from h-monocytes [Livi et al., Mol. Cell. Biol. 10 (1990) 2678-2686]. Our analysis identifies the monocyte clone with r-PDE IVA. Southern blots using a 1.2-kb h-PDE IVB probe at low stringency suggest the presence of additional uncloned human PDE IV family members. Analysis of genomic Southern blots using short specific probes from the h-PDE IVA and h-PDE IVB cDNAs indicates that distinct genes encode these two PDE IV family members. RNA from fractionated normal human leukocytes shows major specific messages of 3.0 and 4.6 kb for h-PDE IVA and 3.7 kb for h-PDE IVB.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , Variación Genética , Familia de Multigenes , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , 3',5'-AMP Cíclico Fosfodiesterasas/química , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Linfocitos B , Secuencia de Bases , Sitios de Unión , Northern Blotting , Southern Blotting , Línea Celular , ADN Recombinante , Escherichia coli , Humanos , Isoenzimas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Procesamiento Proteico-Postraduccional , Pirrolidinonas/farmacología , ARN Mensajero/análisis , Ratas , Proteínas Recombinantes de Fusión/genética , Rolipram , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The effect of mycophenolic acid (MPA), a potent inhibitor of inosine monophosphate dehydrogenase, on the degranulation of rat peritoneal mast cells (RMC) was studied. RMC were pretreated for 48 hr with 0.1-10 microM MPA before the cells were sensitized with IgE and triggered with specific Ag. The net amount of [3H]5-HT released from granules was decreased by 44 and 32% with 1 and 10 microM MPA treatment, respectively. MPA inhibition of degranulation was completely reversed by the addition of 30 microM guanosine to the incubation medium. There was no difference in the apparent number or affinity of IgE binding sites between control and MPA-treated RMC. MPA pretreatment also had no effect on the IgE receptor-mediated production of PGD2 in RMC. These results suggest that depletion of intracellular GTP pools by MPA can disrupt the signaling between the IgE receptor and the secretory granules and that, under these same conditions, the release and metabolism of arachidonic acid are unaffected.
Asunto(s)
Degranulación de la Célula/efectos de los fármacos , IMP Deshidrogenasa/antagonistas & inhibidores , Mastocitos/efectos de los fármacos , Ácido Micofenólico/farmacología , Animales , Sitios de Unión de Anticuerpos , Relación Dosis-Respuesta a Droga , Femenino , Cavidad Peritoneal/citología , Prostaglandina D2/análisis , Ratas , Ratas Endogámicas , Serotonina/análisis , Transducción de Señal/efectos de los fármacosRESUMEN
Arachidonate 15-lipoxygenase (arachidonate:oxygen 15-oxidoreductase, EC 1.13.11.33) is a lipid-peroxidating enzyme that is implicated in oxidizing low density lipoprotein to its atherogenic form. Monocyte/macrophage 15-lipoxygenase is present in human atherosclerotic lesions. To pursue a basis for induction of the enzyme, which is not present in blood monocytes, the ability of relevant cytokines to regulate its expression was investigated. Interleukin 4 (IL-4), among 16 factors tested, specifically induced 15-lipoxygenase mRNA and protein in cultured human monocytes. Interferon gamma and hydrocortisone inhibited this induction. High-performance liquid chromatography analysis of lipid extracts from IL-4-treated monocytes detected 15-lipoxygenase products esterified to the cellular membrane lipids, indicating enzymatic action on endogenous substrates. Stimulation of IL-4-treated monocytes with calcium ionophore or opsonized zymosan A enhanced the formation of 15-lipoxygenase products. These data identify IL-4 and interferon gamma as physiological regulators of lipoxygenase expression and suggest an important link between 15-lipoxygenase function and the immune/inflammatory response in atherosclerosis as well as other diseases.
Asunto(s)
Araquidonato 15-Lipooxigenasa/metabolismo , Inflamación/fisiopatología , Interferón gamma/farmacología , Interleucina-4/farmacología , Monocitos/enzimología , Ácido Araquidónico/metabolismo , Northern Blotting , Citocinas/farmacología , Inducción Enzimática/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Técnicas In Vitro , Lípidos de la Membrana/metabolismo , ARN Mensajero/genéticaRESUMEN
When HL-60 cells are induced to differentiate by dimethyl sulfoxide along a granulocytic pathway there is a fivefold decrease in the total number of transferrin receptors within 3 days, as compared to untreated cells. This decrease is due primarily to a rapid decline in the synthesis of the receptor rather than an increase in the degradation of the receptor. The decrease in transferrin receptor synthesis is a specific and early event that precedes the cessation of cell proliferation, differentiation, and the decrease in total protein synthesis.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Receptores de Transferrina/metabolismo , Línea Celular , Cicloheximida/farmacología , Humanos , Cinética , Leucemia Mieloide Aguda , Receptores de Transferrina/biosíntesis , Receptores de Transferrina/efectos de los fármacosRESUMEN
A mutual antagonism exists between interleukin-1s (IL-1s) as pro-inflammatory and glucocorticoids as anti-inflammatory mediators. This report examines the effects of IL-1 on the induction by dexamethasone of alkaline phosphatase in LEII murine endothelial cells. Dexamethasone increases the specific activity of alkaline phosphatase in a time- and dose-dependent fashion (maximum 14-fold induction at 10(-6) M, IC50 = 10(-8) M), and this induction can be completely inhibited by simultaneous incubation with picomolar concentrations of recombinant human IL-1 alpha or IL-1 beta. This IL-1-mediated antagonism of dexamethasone activity is not due to a down-regulation of glucocorticoid receptors in the cell line used, because the number of receptors and their affinity for dexamethasone is unchanged in IL-1-treated cells. However, induction of alkaline phosphatase by dexamethasone in LEII cells is receptor-mediated, since it can also be inhibited by glucocorticoid-receptor antagonists.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Dexametasona/antagonistas & inhibidores , Endotelio Vascular/enzimología , Interleucina-1/farmacología , Animales , Capilares , División Celular/efectos de los fármacos , Línea Celular , Dexametasona/farmacología , Endotelio Vascular/citología , Inducción Enzimática/efectos de los fármacos , Glucocorticoides/antagonistas & inhibidores , Humanos , Ratones , Receptores de Glucocorticoides/efectos de los fármacos , Proteínas RecombinantesRESUMEN
Monoclonal antibodies (McAb) were developed to the Mr 17,500 form of human recombinant interleukin 1, IL 1 beta. Four McAb have been identified that inhibit the biological activity of IL 1 beta. McAb H34 and H67, at 1 microgram/ml (6 X 10(-9) M), completely inhibit the capacity of 1 ng/ml (6 X 10(-11) M) recombinant IL 1 beta to stimulate the proliferation of murine thymocytes or human fibroblasts in vitro. McAb H6 and H21 are approximately 10-fold less potent, and completely inhibit IL 1 beta activity at 10 micrograms/ml (6 X 10(-8) M) in both assays. The McAb do not have a significant effect on the biological activity of human recombinant IL 1 alpha in either assay. These McAb block the binding of recombinant [125I]IL 1 beta to IL 1 receptors on mouse 3T3 fibroblasts and have affinity constants for IL 1 beta in the range of 10(9) to 10(10) liters/mol. Competition studies suggest that two nonoverlapping epitopes on the IL 1 beta molecule are recognized by the McAb. H6 and H34 recognize one epitope, and H21 and H67 another. McAb H6 and H67 have been used together in a two-site ELISA to detect IL 1 beta. The sensitivity of the ELISA, which is 15 pg/ml (0.86 pM), approaches the limit of sensitivity of the thymocyte proliferation assay. The ELISA and thymocyte proliferation assay were used to quantitate IL 1 beta in E. coli LPS-stimulated human monocyte culture supernatants (HMCS). The level of IL 1 beta detected by ELISA in culture supernatants from eight donors ranged from 1.7 to 5.6 ng/ml, with a mean value of approximately 3 ng/ml. By comparison, the thymocyte proliferation assay gave levels of IL 1 in HMCS that were eight fold higher when quantitated by using recombinant IL 1 beta as a standard. This discrepancy with the bioassay used was reflected by the three fold higher maximum stimulation of thymocyte proliferation by HMCS as compared with recombinant IL 1 alpha or IL 1 beta, and only 45% inhibition of HMCS IL 1 activity by McAb. Thus, factors other than IL 1 beta account for the IL 1-like activity in monocyte culture supernatant as measured by the bioassay. The ILB1 McAb and ELISA allow for the first time-sensitive, accurate, and convenient quantitation of IL 1 beta levels in biological fluids or specimens.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Interleucina-1/inmunología , Animales , Afinidad de Anticuerpos , Epítopos/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Proteínas Recombinantes/inmunologíaRESUMEN
The regulation of alkaline phosphatase activity by steroid hormones was studied in two human breast cancer cell lines, MDA-MB-157 and BT20. MDA-MB-157 cells were shown to express the alkaline phosphatase isoenzyme produced by normal breast tissue, and the activity of this isoenzyme increased 3-fold after a 72-h treatment of these cells with 10(-7) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], 2-fold after treatment with 10(-6) M hydrocortisone (HC), and 5-fold after treatment with both hormones. BT20 cells did not express the isoenzyme phenotypic to breast, but ectopically expressed the isoenzyme phenotypic to term placenta and other embryonic tissue. Treatment of BT20 cells with 1,25-(OH)2D3 results in a 30% decrease in alkaline phosphatase activity of the embryonic isoenzyme. There was a 2-fold increase in activity after treatment with HC, and enzyme activity was similar to control values after treatment with both hormones. For both cell lines, changes in alkaline phosphatase activity correlated with changes in nanograms of isoenzyme per mg cellular protein, as measured by RIA. Increases in enzyme activity were inhibited when the cells were incubated simultaneously with the steroids and cycloheximide. Studies with receptors in each cell line showed that both cell lines bound 1,25-(OH)2D3 and that a 1,25-(OH)2D3-binding protein with the same mol wt as the D3 receptor was present in both. The BT20 cells also express a larger mol wt protein which binds 1,25-(OH)2D3 but is not as specific for the 1,25-(OH)2D3 isomer. HC receptors were similar in quantity and binding affinity in both cell lines.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Calcitriol/fisiología , Isoenzimas/metabolismo , Neoplasias de la Mama/enzimología , Línea Celular , Cicloheximida/farmacología , Dexametasona/farmacología , Humanos , Hidrocortisona/farmacología , Técnicas In Vitro , Radioinmunoensayo , Receptores de Calcitriol , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/metabolismoRESUMEN
The effects of 5-fluorouracil (FUra), 5-fluorouridine (FUrd), and 5-fluoro-2'-deoxyuridine (FdUrd) on L1210 cells were examined in an effort to determine whether the cytotoxicity of these fluoropyrimidines is more closely associated with incorporation of FUra residues into RNA or inhibition of thymidylate (dTMP) synthetase (5,10-methylenetetrahydrofolate: deoxyuridylate C-methyl-transferase, EC 2.1.2.45) by 5-fluoro-2'-deoxyuridylate (FdUMP). In different batches of cells exposed to equitoxic (LD50) doses of these drugs for 48 hr, the levels of free FdUMP, dUMP, and free dTMP synthetase were found to be very similar. However, the number of FUra residues incorporated into total cellular RNA were in the approximate ratio of 1:10:100 in cells treated with FdUrd, FUrd, and FUra, respectively. Although these results are consistent with a common DNA-directed mechanism of toxicity, thymidine (dThd), which should circumvent dTMP synthetase inhibition, did not rescue the cells from the effects of FUra. However, uridine (Urd), which should compete with FUra for incorporation into RNA, had no effect on the toxicity of FUra either. Urd at 10(-5) M did not decrease the amount of incorporation of 10(-7)M [(3)H]FUra into total RNA, but a limited fractionation of polysomal RNA showed about a 4-fold decrease of incorporation of FUra into mRNA in the presence of Urd. Urd and dThd did effectively decrease the cytotoxicity of FUrd and FdUrd, respectively. These observations suggest that cell rescue experiments may not be reliable indicators of the mechanism of cytotoxicity of antimetabolites with complex mechanisms of action.
RESUMEN
The specific activity of alkaline phosphatase was increased in two human osteogenic sarcoma cell lines, SAOS and TE85, after treatment with 1,25 dihydroxy-vitamin D3 (1,25(OH)2D3). Enzyme activity increased when the cells were incubated with concentrations of 1,25(OH)2D3 between 10(-9) and 10(-7) M and cell growth was not inhibited at these concentrations. The specific activity of alkaline phosphatase was 4- to 7-fold higher than that in the control cells after 5 to 7 days of continuous exposure to 1,25(OH)2D3. Immunochemical studies demonstrated that the enzyme from both control and 1,25(OH)2D3-treated cultures cross-reacted with antisera specific for the phosphatase isoenzyme produced by normal human bone, and did not cross-react with antisera specific for the placental alkaline phosphatase isoenzyme. The increased enzyme activity in cultures induced with 1,25(OH)2D3 correlated with an absolute increase in the number of bone-specific phosphatase molecules, as determined by radioimmunoassay. No effect on alkaline phosphatase activity was observed when the cells were treated with other vitamin D metabolites or with 5-bromo-2'-deoxyuridine. Comparative studies demonstrated that hydrocortisone, another steroid hormone, increased the phosphatase activity with a different time course than did 1,25(OH)2D3. High affinity cytoplasmic receptors for 1,25(OH)2D3 and hydrocortisone were found in the SAOS and TE85 cells.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Huesos/enzimología , Calcitriol/farmacología , Isoenzimas/metabolismo , Osteosarcoma/enzimología , Fosfatasa Alcalina/inmunología , Línea Celular , Epítopos/inmunología , Humanos , Hidrocortisona/farmacología , Inmunoensayo , Cinética , Osteosarcoma/metabolismo , Receptores de Calcitriol , Receptores de Glucocorticoides , Receptores de Esteroides/metabolismoRESUMEN
Twenty clones stably resistant to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, or 5-fluorouridine have been isolated from L1210 or P388 murine leukemia cells by a one-step mutation and selection procedure. The activities of enzymes of the pyrimidine salvage pathway relevant to the activation of these drugs have been determined in order to elucidate the mechanisms of resistance in these cells. Cell line resistant to 5-fluorouracil have 7 to 50% of the pyrimidine phosphoribosyltransferase activity found in the wild-type cells, with 5-fluorouracil, uracil, or orotate as substrate. Cells selected for resistance to 5-fluoro-2'-deoxyuridine have no detectable thymidine kinase activity. 5-Fluorouridine-resistant cells have 3 to 25% of the uridine kinase activity measured in the wild-type cell lines. No significant changes were observed in the activities of thymidylate synthetase, nucleoside phosphorylases, or 5-fluorouridylate kinase in any of the resistant cell lines. These findings have relevance to the treatment of human cancer, since pyrimidine phosphoribosyltransferase, thymidine kinase, or uridine kinase could be assayed in tumor biopsies in order to predict whether the fluoropyrimidines would be effective in individual patients.