RESUMO
AIM: We examined the effects of the 11ß-hydroxysteroid dehydrogenase type 1 (HSD1) inhibitor, MK-0916, on the multiple components of the metabolic syndrome (MetS) in patients with type 2 diabetes (T2DM) and MetS. METHODS: This was a 12-week, multicentre, randomized, double-blind, placebo-controlled study. Patients with T2DM (mean baseline A1C: 7.3%) and National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III)-defined MetS were randomized 1 : 1 : 1 : 1 to 0.5, 2 or 6 mg/day MK-0916 or placebo. The primary efficacy endpoint was a change from baseline at week 12 in fasting plasma glucose (FPG). Secondary endpoints included glycosylated haemoglobin A(1c) (A1C), 2-h postprandial glucose (2-h PPG), body weight, waist circumference, blood pressure and lipid profile. RESULTS: Treatment with MK-0916 had no significant effect relative to placebo on FPG at week 12. Compared to placebo, 6 mg MK-0916 produced a modest, significant (p = 0.049) reduction in A1C of 0.3% at week 12, but no significant difference was observed in 2-h PPG. Six milligram MK-0916 increased LDL-C relative to placebo by 10.4% (p = 0.041). Treatment with MK-0916 led to modest dose-dependent decreases in blood pressure and body weight. Overall, MK-0916 was generally well tolerated. MK-0916 produced mechanism-based activation of the hypothalamic-pituitary-adrenal axis, resulting in mean increases in adrenal androgen levels that remained within the normal range at all doses tested. CONCLUSIONS: Inhibition of HSD1 with MK-0916 was generally well tolerated in patients with T2DM and MetS. Although no significant improvement in FPG was observed with MK-0916 compared to placebo, modest improvements in A1C, body weight and blood pressure were observed.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/farmacologia , Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hemoglobinas Glicadas/efeitos dos fármacos , Síndrome Metabólica/tratamento farmacológico , Adolescente , Adulto , Idoso , Análise de Variância , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Masculino , Síndrome Metabólica/metabolismo , Síndrome Metabólica/fisiopatologia , Pessoa de Meia-Idade , Placebos , Período Pós-Prandial , Adulto JovemRESUMO
Pre-receptor metabolism of glucocorticoids by the 11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes has been implicated in the etiology of the metabolic syndrome. Recent studies have shown that alterations in the activity of the type 1 isozyme can affect many aspects of the disease. This paper describes the optimization and application of a high-throughput scintillation proximity assay (SPA) developed to identify selective specific inhibitors of 11betaHSD1. Microsomes containing 11betaHSD1 were incubated in the presence of NADPH and [3H]cortisone, and the product, [3H]cortisol, was specifically detected in the mixture by a monoclonal antibody coupled to protein A-coated SPA beads with greater than 2 log higher affinity for cortisol than cortisone. Dimethyl sulfoxide and NADPH co-substrate additions were optimized for 11betaHSD1 reductase activity. Titrated test compound, when introduced into the optimized assay, reproducibly inhibited the enzyme and yielded consistent IC50 data in either 96- or 384-well format. An 11betaHSD2 counterscreen was performed by incubating 11betaHSD2 microsomes with [3H]cortisol and NAD+ and monitoring substrate disappearance.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Contagem de Cintilação/métodos , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Animais , Anticorpos Monoclonais , Afinidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Dimetil Sulfóxido/farmacologia , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/uso terapêutico , Humanos , Hidrocortisona/análise , Hidrocortisona/imunologia , Hidrocortisona/metabolismo , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/etiologia , Síndrome Metabólica/metabolismo , Microssomos/enzimologia , NADP/metabolismo , Transfecção , TrítioRESUMO
Previous studies have suggested that the terminal complex of complement may contribute to the pathogenesis of atherosclerosis. C5b-9 complexes colocalize with the extracellular lipid in the aortic intima of hypercholesterolemic rabbits, and C6-deficient rabbits develop less atherosclerosis than controls. To test the role of complement in atherosclerosis in a different animal model, C5 deficient (C5def) mice were cross-bred with atherosclerosis susceptible apoE(-/-) mice, generating mice deficient in both apoE and C5 and control apoE(-/-) mice. Progeny were typed for C5 titer and serum cholesterol levels. Both male and female mice were fed a high fat diet from weaning until 22 weeks of age. At that time there were no significant differences in plasma cholesterol or triglycerides between apoE(-/-) control and apoE(-/-)/C5def groups. Morphometric analysis of the aortic root lesions gave mean (+/-SEM) lesion areas for male apoE(-/-) and apoE(-/-)/C5def mice of 468,176 +/- 21,982 and 375,182 +/- 53,089 microm(2), respectively (n = 10 each, P value = 0.123). In female apoE(-/-) mice (n = 5), the mean lesion area was 591,981 +/- 53,242 microm(2), compared to 618,578 +/- 83,457 microm(2) for female apoE(-/-)/C5def mice (n = 10) (P value = 0.835). Thus neither male nor female mice showed a significant change in lesion area when C5 was not present. In contrast to the case in the hypercholesterolemic rabbit, activation of the terminal complex of complement does not play a major role in the development of atherosclerosis in apoE(-/-) mice.
Assuntos
Apolipoproteínas E/fisiologia , Arteriosclerose/genética , Complemento C5/fisiologia , Animais , Aorta/patologia , Apolipoproteínas E/genética , Colesterol/sangue , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Triglicerídeos/sangueRESUMO
11beta-hydroxysteroid dehydrogenases (11beta-HSD) perform prereceptor metabolism of glucocorticoids through interconversion of the active glucocorticoid, cortisol, with inactive cortisone. Although the immunosuppressive and anti-inflammatory activities of glucocorticoids are well documented, the expression of 11beta-HSD enzymes in immune cells is not well understood. Here we demonstrate that 11beta-HSD1, which converts cortisone to cortisol, is expressed only upon differentiation of human monocytes to macrophages. 11beta-HSD1 expression is concomitant with the emergence of peroxisome proliferator activating receptor gamma, which was used as a surrogate marker of monocyte differentiation. The type 2 enzyme, 11beta-HSD2, which converts cortisol to cortisone, was not detectable in either monocytes or cultured macrophages. Incubation of monocytes with IL-4 or IL-13 induced 11beta-HSD1 activity by up to 10-fold. IFN-gamma, a known functional antagonist of IL-4 and IL-13, suppressed the induction of 11beta-HSD1 by these cytokines. THP-1 cells, a human macrophage-like cell line, expressed 11beta-HSD1 and low levels of 11beta-HSD2. The expression of 11beta-HSD1 in these cells is up-regulated 4-fold by LPS. In summary, we have shown strong expression of 11beta-HSD1 in cultured human macrophages and THP-1 cells. The presence of the enzyme in these cells suggests that it may play a role in regulating the immune function of these cells.
Assuntos
Hidroxiesteroide Desidrogenases/biossíntese , Macrófagos/citologia , Macrófagos/enzimologia , Monócitos/citologia , Monócitos/enzimologia , 11-beta-Hidroxiesteroide Desidrogenases , Animais , Calcitriol/farmacologia , Diferenciação Celular/imunologia , Linhagem Celular , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/imunologia , Humanos , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Células U937RESUMO
The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes catalyze the interconversion of active glucocorticoids (GC) with their inert metabolites, thereby regulating the functional activity of GC. While 11beta-HSD type 1 (11beta-HSD1) activates GC from their 11-keto metabolites, 11beta-HSD type 2 (11beta-HSD2) inactivates GC. Here we report that both of these enzymes are expressed in human aortic smooth muscle cells (SMC), and that 11beta-HSD1 is more abundant and is differentially regulated relative to 11beta-HSD2. Stimulation of SMC with IL-1beta or TNFalpha led to a time- and dose-dependent increase of mRNA levels for 11beta-HSD1, while 11beta-HSD2 mRNA levels decreased. Parallel enzyme activity studies showed increased conversion of 3H-cortisone to 3H-cortisol but not 3H-cortisol to 3H-cortisone, demonstrating 11beta-HSD1 in SMC acts primarily as a reductase. A similar increase of 11beta-HSD1 mRNA expression was also found in human bronchial SMC upon stimulation, indicating the regulatory effect is not limited to vascular smooth muscle. Additional parallel studies revealed a similar pattern of induction for 11beta-HSD1 and monocyte chemoattractant protein-1, a well-defined proinflammatory molecule. These data suggest 11beta-HSD1 may play an important role in regulating inflammatory responses in the artery wall and lung.
Assuntos
Aorta/enzimologia , Hidroxiesteroide Desidrogenases/biossíntese , Mediadores da Inflamação/metabolismo , Músculo Liso Vascular/enzimologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2 , Aorta/citologia , Sequência de Bases , Células Cultivadas , Primers do DNA , Indução Enzimática , Humanos , Hidroxiesteroide Desidrogenases/genética , Músculo Liso Vascular/citologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Peroxisome proliferator-activated receptor-gamma (PPARgamma) has been shown to play an important role in the regulation of expression of a subclass of adipocyte genes and to serve as the molecular target of the thiazolidinedione (TZD) and certain non-TZD antidiabetic agents. Hypercorticosteroidism leads to insulin resistance, a variety of metabolic dysfunctions typically seen in diabetes, and hypertrophy of visceral adipose tissue. In adipocytes, the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) converts inactive cortisone into the active glucocorticoid cortisol and thereby plays an important role in regulating the actions of corticosteroids in adipose tissue. Here, we show that both TZD and non-TZD PPARgamma agonists markedly reduced 11beta-HSD-1 gene expression in 3T3-L1 adipocytes. This diminution correlated with a significant decrease in the ability of the adipocytes to convert cortisone to cortisol. The half-maximal inhibition of 11beta-HSD-1 mRNA expression by the TZD, rosiglitazone, occurred at a concentration that was similar to its K(d) for binding PPARgamma and EC(50) for inducing adipocyte differentiation thereby indicating that this action was PPARgamma-dependent. The time required for the inhibitory action of the TZD was markedly greater for 11beta-HSD-1 gene expression than for leptin, suggesting that these genes may be down-regulated by different molecular mechanisms. Furthermore, whereas regulation of PPARgamma-inducible genes such as phosphoenolpyruvate carboxykinase was maintained when cellular protein synthesis was abrogated, PPARgamma agonist inhibition of 11beta-HSD-1 and leptin gene expression was ablated, thereby supporting the conclusion that PPARgamma affects the down-regulation of 11beta-HSD-1 indirectly. Finally, treatment of diabetic db/db mice with rosiglitazone inhibited expression of 11beta-HSD-1 in adipose tissue. This decrease in enzyme expression correlated with a significant decline in plasma corticosterone levels. In sum, these data indicate that some of the beneficial effects of PPARgamma antidiabetic agents may result, at least in part, from the down-regulation of 11beta-HSD-1 expression in adipose tissue.
Assuntos
Adipócitos/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Hidroxiesteroide Desidrogenases/genética , Hidroxiesteroide Desidrogenases/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Tiazóis/farmacologia , Tiazolidinedionas , Fatores de Transcrição/fisiologia , Transcrição Gênica , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1 , Células 3T3 , Adipócitos/enzimologia , Animais , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Cinética , Leptina/genética , Leptina/fisiologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/agonistas , Rosiglitazona , Tiazóis/farmacocinética , Fatores de Transcrição/agonistas , Transcrição Gênica/efeitos dos fármacosRESUMO
Inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, such as simvastatin, lower circulating cholesterol levels and prevent myocardial infarction. Several studies have shown an unexpected effect of HMG-CoA reductase inhibitors on inflammation. Here, we confirm that simvastatin is anti-inflammatory by using a classic model of inflammation: carrageenan-induced foot pad edema. Simvastatin administered orally to mice 1 hour before carrageenan injection significantly reduced the extent of edema. Simvastatin was comparable to indomethacin in this model. To determine whether the anti-inflammatory activity of simvastatin might affect atherogenesis, simvastatin was tested in mice deficient in apoE. Mice were dosed daily for 6 weeks with simvastatin (100 mg/kg body wt). Simvastatin did not alter plasma lipids. Atherosclerosis was quantified through the measurement of aortic cholesterol content. Aortas from control mice (n=20) contained 56+/-4 nmol total cholesterol/mg wet wt tissue, 38+/-2 nmol free cholesterol/mg, and 17+/-2 nmol cholesteryl ester/mg. Simvastatin (n=22) significantly (P<0.02) decreased these 3 parameters by 23%, 19%, and 34%, respectively. Histology of the atherosclerotic lesions showed that simvastatin did not dramatically alter lesion morphology. These data support the hypothesis that simvastatin has antiatherosclerotic activity beyond its plasma cholesterol-lowering activity.
Assuntos
Anti-Inflamatórios/farmacologia , Anticolesterolemiantes/farmacologia , Arteriosclerose/patologia , Arteriosclerose/prevenção & controle , Colesterol/metabolismo , Sinvastatina/metabolismo , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Anticolesterolemiantes/administração & dosagem , Aorta/metabolismo , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Arteriosclerose/sangue , Arteriosclerose/metabolismo , Carragenina/administração & dosagem , Colesterol/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Membro Posterior/patologia , Injeções Subcutâneas , Lipídeos/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sinvastatina/administração & dosagemRESUMO
11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) is an enzyme that converts cortisone to the active glucocorticoid, cortisol. Cortisol-cortisone interconversion plays a key role in the regulation of glucose metabolism, since mice deficient in 11betaHSD1 are resistant to diet-induced hyperglycemia. Peroxisome proliferator activator receptors (PPAR) are key regulators of glucose and lipid homeostasis. We observed a striking downregulation of murine hepatic 11betaHSD1 expression and activity after chronic treatment of wild-type mice with PPARalpha agonists, while 11betaHSD1 in the livers of PPARalpha knockout mice, or in mice treated for only 7 h with PPARalpha agonists, was unaltered. Our results are the first to show PPARalpha agonists can affect glucocorticoid metabolism in the liver by altering 11betaHSD1 expression after chronic treatment. Regulation of active glucocorticoid levels in the liver by PPARalpha agonists may in turn affect glucose metabolism, consistent with reports of their antidiabetic effects.
Assuntos
Fenofibrato/farmacologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Hepatócitos/enzimologia , Hidroxiesteroide Desidrogenases/genética , Fígado/enzimologia , Proliferadores de Peroxissomos/farmacologia , Pirimidinas/farmacologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , 11-beta-Hidroxiesteroide Desidrogenases , Animais , Células Cultivadas , Cricetinae , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retroalimentação , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Knockout , Modelos Biológicos , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/agonistas , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)-/- mice that were also deficient in iNOS. ApoE-/- and iNOS-/- mice were cross-bred to produce apoE-/-/iNOS-/- mice and apoE-/-/iNOS+/+ controls. Males and females were placed on a high fat diet at the time of weaning, and atherosclerosis was evaluated at two time points by different methods. The deficiency in iNOS had no effect on plasma cholesterol, triglyceride, or nitrate levels. Morphometric measurement of lesion area in the aortic root at 16 wk showed a 30-50% reduction in apoE-/-/iNOS-/- mice compared with apoE-/-/iNOS+/+ mice. Although the size of the lesions in apoE-/-/iNOS-/- mice was reduced, the lesions maintained a ratio of fibrotic:foam cell-rich:necrotic areas that was similar to controls. Biochemical measurements of aortic cholesterol in additional groups of mice at 22 wk revealed significant 45-70% reductions in both male and female apoE-/-/iNOS-/- mice compared with control mice. The results indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion.
Assuntos
Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Arteriosclerose/enzimologia , Arteriosclerose/genética , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Animais , Aorta/enzimologia , Aorta/metabolismo , Arteriosclerose/sangue , Arteriosclerose/patologia , Colesterol/sangue , Colesterol/metabolismo , Feminino , Predisposição Genética para Doença/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitratos/sangue , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Triglicerídeos/sangueRESUMO
LRP5 is a novel member of the low-density lipoprotein receptor family that is genetically associated with Type 1 diabetes. As a start to defining the normal function of LRP5 and to generate testable hypotheses of its potential role in Type 1 diabetes pathogenesis, we carried out an extensive expression analysis of this gene at the mRNA and protein levels in normal human, monkey, and mouse, as well as in non-obese diabetic (NOD) mice at several stages of diabetes development. In all species, expression of LRP5 was found in four functionally important cell types: the distributed mononuclear phagocyte system, the islets of Langerhans, vitamin A-metabolizing cells, and CNS neurons. Given the critical role of macrophages in the onset and progression of islet cell destruction in Type 1 diabetes and the hypothesized role of retinoids as modifiers of diabetes progression, these findings suggest that LRP5 may confer Type 1 diabetes risk by altering the normal functioning of one or more of these regulatory systems. Specifically, given that the LRP5 polymorphisms associated with diabetes are in the promoter region of the gene, alterations in LRP5 expression may be responsible for diabetes susceptibility and therefore may be potential targets for therapeutic intervention. (J Histochem Cytochem 48:1357-1368, 2000)
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Ilhotas Pancreáticas/metabolismo , Macrófagos/metabolismo , Receptores de LDL/metabolismo , Vitamina A/metabolismo , Animais , Western Blotting , Linhagem Celular , Chlorocebus aethiops , Humanos , Imuno-Histoquímica , Hibridização In Situ , Túbulos Renais/metabolismo , Proteínas Relacionadas a Receptor de LDL , Fígado/citologia , Fígado/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Macaca mulatta , Camundongos , Camundongos Endogâmicos NOD , Neurônios/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Baço/citologia , Baço/metabolismo , Timo/citologia , Timo/metabolismoRESUMO
Recent work has revealed correlations between bacterial or viral infections and atherosclerotic disease. One particular bacterium, Chlamydia pneumoniae, has been observed at high frequency in human atherosclerotic lesions, prompting the hypothesis that infectious agents may be necessary for the initiation or progression of atherosclerosis. To determine if responses to gram-negative bacteria are necessary for atherogenesis, we first bred atherosclerosis-prone apolipoprotein (apo) E(-/)- (deficient) mice with animals incapable of responding to bacterial lipopolysaccharide. Atherogenesis was unaffected in doubly deficient animals. We further tested the role of infectious agents by creating a colony of germ-free apo E(-/)- mice. These animals are free of all microbial agents (bacterial, viral, and fungal). Atherosclerosis in germ-free animals was not measurably different from that in animals raised with ambient levels of microbial challenge. These studies show that infection is not necessary for murine atherosclerosis and that, unlike peptic ulcer, Koch's postulates cannot be fulfilled for any infectious agent in atherosclerosis.
Assuntos
Arteriosclerose/etiologia , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Arteriosclerose/patologia , Chlamydophila pneumoniae/patogenicidade , Vida Livre de Germes , Humanos , Infecções/complicações , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
Macrophage migration inhibitory factor (MIF) is a cytokine that was first described as an inhibitor of the random migration of monocytes and macrophages and has since been proposed to have a number of immune and catalytic functions. One of the functions assigned to MIF is that of a tautomerase that interconverts the enol and keto forms of phenylpyruvate and (p-hydroxyphenyl)pyruvate and converts D-dopachrome, a stereoisomer of naturally occurring L-dopachrome, to 5,6-dihydroxyindole-2-carboxylic acid. The physiological significance of the MIF enzymatic activity is unclear. The three-dimensional structure of MIF is strikingly similar to that of two microbial enzymes (4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase) that otherwise share little sequence identity with MIF. MIF and these two enzymes have an invariant N-terminal proline that serves as a catalytic base. Here we report a new biological function for MIF, as an inhibitor of monocyte chemoattractant protein 1- (MCP-1-) induced chemotaxis of human peripheral blood monocytes. We find that MIF inhibition of chemotaxis does not occur at the level of the CC chemokine receptor for MCP-1, CCR2, since MIF does not alter the binding of (125)I-MCP-1 to monocytes. The role of MIF enzymatic activity in inhibition of monocyte chemotaxis and random migration was studied with two MIF mutants in which the N-terminal proline was replaced with either a serine or a phenylalanine. Both mutants remain capable of inhibiting monocyte chemotaxis and random migration despite significantly reduced or no phenylpyruvate tautomerase activity. These data suggest that this enzymatic activity of MIF does not play a role in its migration inhibiting properties.
Assuntos
Movimento Celular/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Fatores Inibidores da Migração de Macrófagos/farmacologia , Receptores de Quimiocinas , Animais , Sequência de Bases , Quimiocina CCL2/metabolismo , Dicroísmo Circular , Primers do DNA , Humanos , Técnicas In Vitro , Cinética , Espectrometria de Massas , Camundongos , Receptores CCR2 , Receptores de Citocinas/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Intraepithelial lymphocytes (IEL) are a phenotypically distinct population of lymphocytes that reside in mucosal epithelia, below the intercellular tight junctions. Although adhesive functions of this population have been previously studied, relatively little is known about IEL migration from the microvasculature into the epithelium. We demonstrated that cultured human IEL were capable of migration into polarized epithelial cells in vitro, where they assumed a subjunctional position, identical to that observed in vivo. The migration was rapid and efficient and was directionally polarized, such that IEL migrated into epithelial monolayers from the basolateral, but not the apical, aspect. After a 4-h period of residence, up to one-half of the IEL then exited the monolayer basolaterally. Migration was partially inhibited by pertussis toxin, suggesting a potential mechanism for IEL migration by chemokine receptor-mediated signaling. The conditions and ligand pairs used in IEL migration were different from those for neutrophils, another cell type known to migrate through epithelia. This system may serve as a model for microenvironmental homing of IEL into the epithelium.
Assuntos
Mucosa Intestinal/fisiologia , Linfócitos/fisiologia , Neutrófilos/fisiologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/fisiologia , Linhagem Celular , Movimento Celular , Polaridade Celular , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/fisiologia , Humanos , Integrinas/biossíntese , Integrinas/fisiologia , Mucosa Intestinal/imunologia , Cinética , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Proteínas Recombinantes/biossíntese , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Fatores de Tempo , TransfecçãoRESUMO
Therapy-related acute myelogenous leukemia and myelodysplastic syndrome (t-AML/MDS) are being reported with increasing frequency as a complication of ABMT for Hodgkin's disease and non-Hodgkin's lymphoma. At present there is no method available to predict who is at risk or is destined to develop this nearly universally fatal disorder. We therefore investigated whether clonal growth of cells is predictive of the development of t-AML/MDS. In a patient who developed secondary AML/MDS 18 months after ABMT, X-linked clonality analysis at the human androgen receptor locus was performed on serial banked samples, and documented transition from polyclonal to clonal hematopoiesis. Clonal cells could be identified 6 months after transplant (1 year prior to the diagnosis of t-AML/MDS), at a time when there was no morphologic or clinical evidence of disease. Clonality analysis can be predictive of the development of t-AML/MDS after ABMT and may offer important insights into associated risk factors and strategies to minimize the risk of t-AML/MDS.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Leucemia Mieloide Aguda/genética , Linfoma/terapia , Síndromes Mielodisplásicas/genética , Receptores Androgênicos/genética , Alelos , Feminino , Hematopoese/genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/etiologia , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/etiologia , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Transplante Autólogo , Cromossomo XRESUMO
Caveolae are 50-100-nm membrane microdomains that represent a subcompartment of the plasma membrane. Previous morphological studies have implicated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial cells, (b) the uptake of small molecules via a process termed potocytosis involving GPI-linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartmentalization of certain signaling molecules, including G-protein coupled receptors. Caveolin, a 22-kD integral membrane protein, is an important structural component of caveolae that was first identified as a major v-Src substrate in Rous sarcoma virus transformed cells. This finding initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation. We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured cells. To facilitate biochemical manipulations, we have applied this procedure to lung tissue--an endothelial and caveolin-rich source-allowing large scale preparation of these complexes. These membrane domains retain approximately 85% of caveolin and approximately 55% of a GPI-linked marker protein, while they exclude > or = 98% of integral plasma membrane protein markers and > or = 99.6% of other organelle-specific membrane markers tested. Characterization of these complexes by micro-sequencing and immuno-blotting reveals known receptors for modified forms of LDL (scavenger receptors: CD 36 and RAGE), multiple GPI-linked proteins, an anion transporter (plasma membrane porin), cytoskeletal elements, and cytoplasmic signaling molecules--including Src-like kinases, hetero-trimeric G-proteins, and three members of the Rap family of small GTPases (Rap 1--the Ras tumor suppressor protein, Rap 2, and TC21). At least a fraction of the actin in these complexes appeared monomeric (G-actin), suggesting that these domains could represent membrane bound sites for microfilament nucleation/assembly during signaling. Given that the majority of these proteins are known molecules, our current studies provide a systematic basis for evaluating these interactions in vivo.
Assuntos
Caveolinas , Endotélio Vascular/química , Membranas Intracelulares/química , Pulmão/química , Proteínas de Membrana/química , Sequência de Aminoácidos , Animais , Antígenos CD/análise , Antígenos CD36 , Caveolina 1 , Compartimento Celular , Transformação Celular Viral , Membranas Intracelulares/ultraestrutura , Camundongos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Análise de Sequência , Transdução de Sinais , Frações Subcelulares/química , Distribuição TecidualRESUMO
The avidity of integrin CR3 (also known as alpha M beta 2, Mac-1, Mo-1, and CD11b/CD18) may be reversibly altered without changes in the number of cell surface receptors. Here we describe a molecule termed integrin modulating factor (IMF-1), which controls CR3 avidity. Addition of IMF-1 to polymorphonuclear leukocytes (PMNs) or to purified CR3 causes enhanced binding of ligand. IMF-1 is not present in resting PMNs, but stimulation of cells results in a transient rise in IMF-1 content that parallels a transient rise in CR3 activity. We suggest that PMNs control adhesivity by controlling synthesis of IMF-1, which then acts as an allosteric activator of leukocyte integrins. IMF-1 is an acidic, amphiphilic molecule of Mr340 +/- 16 that does not contain ester, phosphate, amide, sialic acid, or glycosidic or vicinal hydroxyl functionalities, but does contain a carbon-carbon double bond. These results suggest that IMF-1 is an unsaturated fatty acid or an isoprenoid acid.
Assuntos
Adesão Celular/fisiologia , Ácidos Graxos Insaturados/fisiologia , Ácidos Graxos/fisiologia , Antígeno de Macrófago 1/fisiologia , Neutrófilos/fisiologia , Adesão Celular/efeitos dos fármacos , Ácidos Graxos/química , Ácidos Graxos Insaturados/química , Citometria de Fluxo , Cinética , Antígeno de Macrófago 1/química , Antígeno de Macrófago 1/efeitos dos fármacos , Antígeno de Macrófago 1/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Tumor necrosis factor alpha, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, and formyl peptide were each found to cause a twofold increase in expression of CD14 on the surface of polymorphonuclear leukocytes (PMN). Upregulation of CD14 was complete by 20 min and thus appeared to result from expression of preformed stores of protein. The CD14 on the surface of PMN was shown to serve two biological functions. It bound particles coated with complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP). This binding activity was enhanced by agonists that upregulated CD14 expression and may serve in the clearance of Gram-negative bacteria opsonized with LBP. Interaction of CD14 with LPS in the presence of LBP or serum also caused a dramatic, transient increase in the adhesive activity of CR3 (CD11b/CD18) on PMN. Enhanced activity of CR3 and other members of the CD11/CD18 family underlies many of the known physiological responses of PMN to LPS and may be a central feature of the in vivo responses of PMN to endotoxin.
Assuntos
Proteínas de Fase Aguda , Antígenos de Diferenciação Mielomonocítica/fisiologia , Proteínas de Transporte/fisiologia , Ativação do Complemento/fisiologia , Endotoxinas/farmacologia , Antígeno de Macrófago 1/efeitos dos fármacos , Glicoproteínas de Membrana , Neutrófilos/ultraestrutura , Antígenos de Diferenciação Mielomonocítica/metabolismo , Proteínas de Transporte/metabolismo , Ativação do Complemento/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Receptores de Lipopolissacarídeos , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/fisiologia , Antígeno de Macrófago 1/metabolismo , Antígeno de Macrófago 1/fisiologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The c-fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v-fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c-fos is a member of a family of structurally related proteins which includes the fos-related antigens (fra). We have studied the dynamic state of the c-fos protein with an antibody prepared by immunizing rabbits with a plasmid-encoded fos fusion protein. In serum-stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS-PAGE as a 60-68-kD group of bands corresponding to c-fos, a doublet at 44-45-kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50-kD band corresponding to a fra. We show that although c-fos protein synthesis is only transiently induced by serum, the c-fos protein persists within the cell after its synthesis has ceased, and it decays with a half-life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune-precipitated complex even at times when c-fos is no longer synthesized. These kinetics indicate that even following shutoff of c-fos protein synthesis, p39 is newly synthesized and can complex with c-fos protein or a fos-related antigen. During this time, c-fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c-fos and/or the fos-related antigens over a period of 4-8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c-fos and fra protein interactions for the function of these proteins.
Assuntos
Ciclo Celular , Proteínas Proto-Oncogênicas/metabolismo , Animais , Especificidade de Anticorpos , Western Blotting , Linhagem Celular , Regulação da Expressão Gênica , Técnicas Imunológicas , Substâncias Macromoleculares , Camundongos , Fosfoproteínas/fisiologia , Testes de Precipitina , Proteínas Proto-Oncogênicas c-fos , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Human leukocytes express a receptor that mediates the binding of cells and particles coated with C3bi, a fragment of the third component of complement. Previous data indicate that the capacity of this receptor to mediate binding is regulated by changes in its aggregation state. Randomly distributed receptors bind ligand very inefficiently, but stimulation of polymorphonuclear leukocytes with phorbol esters causes a ligand-independent clustering of the receptors in the membrane, and the clustered receptors avidly bind C3bi-coated cells (1). We examined whether the aggregation state of surface-bound ligands also affects the efficiency of binding between receptors and ligands. We found that erythrocytes bearing C3bi in clusters were bound by both macrophages and polymorphonuclear leukocytes far more avidly than erythrocytes bearing the same number of ligands in random array. We made similar observations with erythrocytes coated with C3b, a ligand that is recognized by a separate receptor. Our observations show that the ability of a receptor-bearing cell to bind particles coated with the corresponding ligands is dramatically affected by the distribution of ligand on the surface of the particle. Cell-cell interactions may thus be regulated by alterations in the two-dimensional distribution of receptors and ligands on opposing cell surfaces.