RESUMEN

Human monocyte chemoattractant protein-1 (MCP-1) is expressed by a variety of cell types in response to various stimuli. MCP-1 expressed by the endothelium plays an important role in cell migration and activation. MCP-1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we present evidence that the proteasome complex is involved in mediating the interleukin (IL)-1beta induction of MCP-1 in endothelial cells. We present evidence that a proteasome inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal (norLeu), and the protease inhibitor tosyl-Phe-chloromethylketone (TPCK) block IL-1beta induction of MCP-1 protein expression. norLeu and TPCK also blocked IL-1beta-induced MCP-1 promoter-driven reporter gene expression as well as nuclear factor (NF)-kappaB-mediated reporter gene expression. The effects of norLeu were due to its inhibition of the proteasome rather than calpain, because other calpain inhibitors had no effect on MCP-1 expression. In contrast to TPCK, which blocked NF-kappaB translocation to the nucleus, norLeu had no effect on NF-kappaB nuclear translocation or IL-1beta-induced phosphorylation of p65. This study demonstrates that the proteasome pathway is involved in IL-1beta-induced MCP-1 gene expression in human endothelial cells.

Asunto(s)

Quimiocina CCL2/genética , Cisteína Endopeptidasas/metabolismo , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Interleucina-1/fisiología , Complejos Multienzimáticos/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Leupeptinas/farmacología , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal , Clorometilcetona de Tosilfenilalanila/farmacología

RESUMEN

Chemokines are potent mediators of cell migration and activation and therefore play an essential role in early events of inflammation. In conjunction with cell adhesion molecules, chemokines help to localize cells to a specific site and enhance the inflammatory reaction at the site. Clinically, elevated levels of chemokines have been found in a variety of inflammatory diseases. The prototype C-C chemokine is monocyte chemoattractant protein-1 (MCP-1) which is synthesized by variety of cell types including endothelial cells in response to a variety of stimuli. MCP-1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we investigated the factors involved in cytokine-induced MCP-1 gene expression in human endothelial cells. We present evidence that the nuclear factor (NF)-kappa B-like binding site and the AP-1 binding site located 90 and 68 base pairs upstream of the transcriptional start site, respectively, are required for maximal induction of the human MCP-1 promoter by interleukin-(IL)-1 beta. Site-directed mutagenesis or deletion of the NF-kappa B-like site decreased the cytokine-induced activity of the promoter. Site-directed mutagenesis of the AP-1 binding site also decreased the cytokine-induced activity of the promoter. We show that the NF-kappa B-like site located at-90 in the MCP-1 promoter binds to the p50/p65 heterodimer of the NF-kappa B/Rel family in IL-1 beta-stimulated human endothelial cells. Overexpression of p65 results in the transactivation of the MCP-1 promoter as well. The data presented in this study suggest that cytokine-induced MCP-1 gene expression in human endothelial cells depends on the cooperative action of NF-kappa B and AP-1.

Asunto(s)

Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Citocinas/fisiología , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/inmunología , FN-kappa B/fisiología , Factor de Transcripción AP-1/fisiología , Secuencia de Bases , Células Cultivadas , Dimerización , Humanos , Interleucina-1/fisiología , Datos de Secuencia Molecular , FN-kappa B/biosíntesis , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B , Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-rel , Factor de Transcripción ReIA , Factores de Transcripción/biosíntesis , Factor de Necrosis Tumoral alfa/fisiología , Venas Umbilicales/citología

RESUMEN

Inflammation is characterized by the recruitment of leukocytes and their subsequent migration from the vasculature into the tissue, where they often cause severe damage. Endothelial cells play a major role in this cascade by expressing cell surface adhesion molecules, such as VCAM-1 and ICAM-1, and chemokines, in response to cytokines. Many of these genes are under the control of inflammatory response transcription factors such as nuclear factor (NF)-kappa B. In this study, we examined the effects of 5-lipoxygenase inhibitors (nordihydroguaiaretic acid and AA861) on IL-1 beta-induced VCAM-1 gene expression in HUVECs. We demonstrated that 5-lipoxygenase inhibitors, but not cyclooxygenase inhibitors, block IL-1 beta-induced VCAM-1 cell surface expression and promoter activity. In transiently transfected HUVECs, NF-kappa B-dependent gene expression was inhibited by 5-lipoxygenase inhibitors. These inhibitors did not block IL-1 beta-induced nuclear translocation of NF-kappa B, inhibitor of kappa B-alpha proteolytic degradation, or significantly reduce phosphorylation of p65. These studies indicate that inhibition of 5-lipoxygenase blocks cytokine-induced VCAM-1 gene expression by reducing the functional activity of NF-kappa B/Rel proteins in HUVECs.

Asunto(s)

Endotelio Vascular/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , Inhibidores de la Lipooxigenasa , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/genética , Benzoquinonas/farmacología , Transporte Biológico/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Inhibidores de la Lipooxigenasa/farmacología , Masoprocol/farmacología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/efectos de los fármacos

RESUMEN

At sites of inflammation, endothelial cells play a major role in defining the types of leukocytes that are recruited to a specific area. This is accomplished, at least in part, through the cytokine induction of cell surface adhesion molecules, including vascular cell adhesion molecule 1 (VCAM-1). We investigated the role of phosphatidylcholine-specific phospholipase C in the induction of VCAM-1 gene expression by interleukin-1 beta. D609, a phosphatidylcholine-specific phospholipase C inhibitor, reduced VCAM-1 cell surface expression and VCAM-1 promoter activity in human endothelial cells in a dose-dependent manner. D609 did not affect nuclear translocation of nuclear factor-kappa B but inhibited nuclear factor-kappa B-mediated transcription. The results of this study indicate that phosphatidylcholine-specific phospholipase C is required for activation of nuclear factor-kappa B and cytokine induction of VCAM-1 gene expression in endothelial cells.

Asunto(s)

Hidrocarburos Aromáticos con Puentes/farmacología , Endotelio Vascular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , Tionas/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Molécula 1 de Adhesión Celular Vascular/genética , Secuencia de Bases , Células Cultivadas , Endotelio Vascular/metabolismo , Humanos , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Norbornanos , Regiones Promotoras Genéticas , Tiocarbamatos , Fosfolipasas de Tipo C/fisiología

RESUMEN

Endothelial cells play a major role in recruiting leukocytes to sites of inflammation. This is accomplished, at least in part, by up-regulation of cell surface adhesion molecules, including VCAM-1 and ICAM-1, in response to cytokines. In this report, we investigated the role of the proteasome complex in mediating the interleukin (IL)- 1 beta induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells. We present evidence that a proteasome inhibitor, n-acetyl-leucinyl-leucinyl-norleucinal (norLEU), as well as specific protease inhibitors, n-tosyl-Lys-chloromethylketone and N-tosyl-Phe-chloromethylketone, blocked IL-1 beta induction of VCAM-1 and ICAM-1 promoter-driven reporter gene expression in stably transfected endothelial cells. These inhibitors also blocked cytokine induced cell surface expression of VCAM-1 and ICAM-1 by human umbilical vein endothelial cells. As expected, the protease inhibitors blocked the activation of nuclear factor (NF)-kappa B in response to IL-1 beta stimulation. In contrast, norLEU did not prevent IL-1 beta-induced nuclear translocation of NF-kappa B. The effects of norLEU were specific because it did not inhibit the IL-1 beta induction of plasminogen activator inhibitor type 1 gene expression. This study demonstrates that inhibition of the proteolytic activity of the proteasome blocks IL-1 beta induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells.

Asunto(s)

Calpaína/fisiología , Núcleo Celular/metabolismo , Cisteína Endopeptidasas/fisiología , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/biosíntesis , Leupeptinas/farmacología , Complejos Multienzimáticos/fisiología , FN-kappa B/metabolismo , Clorometilcetona Tosilisina/farmacología , Clorometilcetona de Tosilfenilalanila/farmacología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Secuencia de Bases , Transporte Biológico/efectos de los fármacos , Calpaína/antagonistas & inhibidores , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/genética , Interleucina-1/farmacología , Datos de Secuencia Molecular , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Molécula 1 de Adhesión Celular Vascular/genética

RESUMEN

Vascular endothelial cells respond to cytokines such as IL-1 beta or TNF-alpha by undergoing a number of functional alterations. Among these alterations is the induction of cell surface adhesion molecules, including VCAM-1. In this report, we investigated the effects of a 3-alkoxybenzo[beta]thiophene-2-carboxamide (BZT) on the cytokine induction of VCAM-1 expression and activation of the transcription factor NF-kappa B in human endothelial cells. BZT blocked the IL-1 beta induced cell surface expression of VCAM-1 in human endothelial cells but did not prevent nuclear translocation of NF-kappa B. This study demonstrates that BZT is a potent inhibitor of VCAM-1 expression in human endothelial cells.

Asunto(s)

Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , FN-kappa B/metabolismo , Tiofenos/farmacología , Molécula 1 de Adhesión Celular Vascular/genética , Secuencia de Bases , Transporte Biológico , Núcleo Celular/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Tiofenos/síntesis química , Clorometilcetona de Tosilfenilalanila/farmacología , Venas Umbilicales

RESUMEN

A general solid-phase method for the site-directed mutagenesis of double-stranded DNA (dsDNA) is described. Plasmid DNA is linearized using either a restriction endonuclease (ENase) or the RecA-assisted ENase or RecA-AC cleavage method. Alternatively, PCR may be used to generate linear dsDNA. One or both strands of the DNA is biotinylated and attached to a solid support, and the DNA strands are separated using 0.2 M NaOH. An extension oligodeoxyribonucleotide (oligo) and a single or multiple oligo(s) containing the desired mutation(s) are annealed to one of the bound DNA strands and used to initiate the synthesis of a complementary strand by a nonstrand-displacing DNA polymerase. The in vitro synthesized strand incorporating the desired alteration(s) is melted off of the support and recircularized using one of several types of bridging oligos, DNA ligase, and a DNA polymerase and transformed into the host. Greater than 90% mutagenic efficiency has been obtained using this method.

Asunto(s)

ADN/genética , Mutagénesis Sitio-Dirigida , Secuencia de Bases , Biotina/metabolismo , ADN Circular , Escherichia coli/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Transformación Bacteriana

RESUMEN

Transgenic mice were constructed using human alpha 1-antitrypsin M and Z genomic clones. Livers of the M lineage mice showed slight cellular pleomorphism and immunohistochemically demonstrable finely granular alpha 1-antitrypsin material in hepatocytes. Z lineage mice with five gene copies per haploid mouse genome (Z#1) demonstrated fine granular alpha 1-antitrypsin material and a few large globules. In contrast, Z lineage mice with 12 gene copies per haploid mouse genome (Z#2) demonstrated hepatocytes filled with homogeneous, eosinophilic globules that were strongly reactive with diastase and periodic acid-Schiff and antibody to alpha 1-antitrypsin. Scattered microscopic polymorphonuclear leukocyte accumulations were seen that contained extracellular alpha 1-antitrypsin material, but there was neither histological nor serological evidence of mouse infectious hepatitis. In young animals, small clusters of hepatocytes lacking alpha 1-antitrypsin material were seen. These cells were the dominant population in older animals and formed nodular arrangements. Fibrosis was not demonstrable in neonatal and young animals or in any of the controls, but perisinusoidal fibrosis was seen in older Z#2 mice. Groups of hepatocytes without alpha 1-antitrypsin material showed dysplastic changes. We conclude that the transgenic mouse is a reliable and useful model in which to study the effects of alpha 1-antitrypsin in the liver because it demonstrates changes similar to those in the human disease.

Asunto(s)

Hepatopatías/patología , Hígado/patología , alfa 1-Antitripsina/genética , Alelos , Animales , Ensayo de Inmunoadsorción Enzimática , Humanos , Hepatopatías/genética , Ratones , Ratones Transgénicos , ARN/genética , ARN/aislamiento & purificación , Valores de Referencia