RESUMEN
Factors that modulate cholesterol levels have major impacts on cardiovascular disease. Niemann-Pick C1-like 1 (NPC1L1) functions as a sterol transporter mediating intestinal cholesterol absorption and counter-balancing hepatobiliary cholesterol excretion. The liver receptor homolog 1 (LRH-1) had been shown to regulate genes involved in hepatic lipid metabolism and reverse cholesterol transport. To study whether human NPC1L1 gene is regulated transcriptionally by LRH-1, we have analyzed evolutionary conserved regions (ECRs) in HepG2 cells. One ECR was found to be responsive to the LRH-1. Through deletion studies, LRH-1 response element was identified and the binding of LRH-1 was demonstrated by EMSA and ChIP assays. When SREBP2, one of several transcription factors which had been shown to regulate NPC1L1 gene, was co-expressed with LRH-1, synergistic transcriptional activation resulted. In conclusion, we have identified LRH-1 response elements in NPC1L1 gene and propose that LRH-1 and SREBP may play important roles in regulating NPC1L1 gene.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de la Membrana/genética , Colesterol/metabolismo , Secuencia Conservada , Células Hep G2 , Humanos , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Elementos Reguladores de la Transcripción , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
The ATP-binding cassette transporters ABCG5 and ABCG8 form heterodimers that limit absorption of dietary sterols in the intestine and promote cholesterol elimination from the body through hepatobiliary secretion. To identify cis-regulatory elements of the two genes, we have cloned and analyzed twenty-three evolutionary conserved region (ECR) fragments using the CMV-luciferase reporter system in HepG2 cells. Two ECRs were found to be responsive to the Liver-X-Receptor (LXR). Through elaborate deletion studies, regions containing putative LXREs were identified and the binding of LXRα was demonstrated by EMSA and ChIP assay. When the LXREs were inserted upstream of the intergenic promoter, synergistic activation by LXRα/RXRα in combination with GATA4, HNF4α, and LRH-1, which had been shown to bind to the intergenic region, was observed. In conclusion, we have identified two LXREs in ABCG5/ABCG8 genes for the first time and propose that these LXREs, especially in the ECR20, play major roles in regulating these genes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Lipoproteínas/genética , Receptores Nucleares Huérfanos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5 , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Transportadoras de Casetes de Unión a ATP/metabolismo , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Células Hep G2 , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Lipoproteínas/metabolismo , Receptores X del Hígado , Receptores Nucleares Huérfanos/genética , Regiones Promotoras Genéticas , Unión Proteica , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Elementos Reguladores de la Transcripción/genética , Elementos de Respuesta/genética , Activación TranscripcionalRESUMEN
Heat shock protein 70 (Hsp70) release and its effects on pro-inflammatory cytokine production have been controversial. In this study, we investigated whether Hsp70 could be released from monocytes and activates matrix metalloproteinase-9 (MMP-9) gene expression. Hsp70 overexpression in human monocytic cell line U937 was found to increase PMA-induced MMP-9 expression and enhance cell motility. Hsp70 cDNA transfectants released Hsp70 protein into culture supernatants, and a part of released Hsp70 subsequently was bound to the surface of U937 cells. Addition of culture medium containing the extracelluar Hsp70 led to an increase not only in proMMP-9 secretion, but also the invasiveness of U937 cells through Matrigel or human umbilical vascular endothelial cells (HUVEC) in vitro. Immunodepletion of Hsp70 abolished its effect on MMP-9 expression. The released Hsp70 activated nuclear factor kappa B (NF-kappaB) and activating protein-1 (AP-1), which led to the activation of MMP-9 transcription. Taken together, these results suggest that extracellular Hsp70 induces the expression of MMP-9 gene through activation of NF-KappaB and AP-1.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , Movimiento Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Regulación de la Expresión Génica , Humanos , FN-kappa B/metabolismo , Factor de Transcripción AP-1/metabolismo , Transfección , Células U937RESUMEN
The Drosophila methuselah (mth) mutant has an approximately 35 percent increase in average lifespan, and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat. To examine the transcriptional regulation of mth, we used luciferase assays employing Drosophila S2 cells. Two positive control elements were found at -542 to -272 (PE1) and +28 to +217 (PE2), where putative binding sites for transcription factors including Dorsal (Dl) were identified. Cotransfection of a Dl expression plasmid with a mth-luciferase reporter plasmid resulted in decreased reporter activity. PE1 and PE2, the minimal elements for strong promoter activity, were required for maximal repression by Dl protein. The N-terminal Rel homology domain (RHD) of Dl was not sufficient for repression of mth. We demonstrated by chromatin affinity precipitation (ChAP) assays in S2 cells that Dl bound to the putative PE1 binding site. Unexpectedly, semi-quantitative RT-PCR analysis revealed that the level of mth transcripts was reduced in dl flies. However, the in vivo result support the view that mth expression is regulated by dl, since it is well known that Dl functions as both a transcriptional activator and repressor depending on what other transcription factors are present. These findings suggest that both innate immunity and resistance to stress are controlled by Dl protein.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Genes Reporteros , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Receptores Acoplados a Proteínas G/genética , Análisis de Secuencia de ADN , Factores de Transcripción/genéticaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD) is responsible for highly efficient protein transduction across plasma membranes. In a previous study, we showed that Tat-Cu,Zn-superoxide dismutase (Tat-SOD) can be directly transduced into mammalian cells across the lipid membrane barrier. In this study, we fused the human SOD gene with a Tat PTD transduction vector at its N- and/or C-terminus. The fusion proteins (Tat-SOD, SOD-Tat, Tat-SOD-Tat) were purified from Escherichia coli and their ability to enter cells in vitro and in vivo compared by Western blotting and immunohistochemistry. The transduction efficiencies and biological activities of the SOD fusion protein with the Tat PTD at either terminus were equivalent and lower than the fusion protein with the Tat PTD at both termini. The availability of a more efficient SOD fusion protein provides a powerful vehicle for therapy in human diseases related to this anti-oxidant enzyme and to reactive oxygen species.
Asunto(s)
Productos del Gen tat/metabolismo , Piel/metabolismo , Superóxido Dismutasa/metabolismo , Transducción Genética , Animales , Supervivencia Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Células HeLa , Humanos , Ratones , Plásmidos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Piel/citología , Superóxido Dismutasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Recent studies in our laboratory demonstrate that ligand-mediated activation of the EphA8 receptor critically regulates cell adhesion and migration. In this report, we show that the EphA8 receptor induces neurite outgrowth in NG108-15 cells in the absence of ligand stimulation. Using various deletion mutants lacking specific intracytoplasmic regions, we confirm that the tyrosine kinase domain of EphA8 is important for inducing neurite outgrowth. However, the tyrosine kinase activity of EphA8 is not crucial for neurite outgrowth induction. Treatment with various inhibitors further reveals that the mitogen-activated protein kinase (MAPK) signaling pathway is critical for neurite outgrowth induced by EphA8. Consistent with these results, EphA8 expression induced a sustained increase in the activity of MAPK, whereas ligand-mediated EphA8 activation had no further modulatory effects on MAP kinase activity. Additionally, activated MAPK relocalized from the cytoplasm to the nucleus in response to EphA8 transfection. These results collectively suggest that the EphA8 receptor is capable of inducing a sustained increase in MAPK activity, thereby promoting neurite outgrowth in neuronal cells.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/farmacología , Neuritas , Receptor EphA8/genética , Receptor EphA8/fisiología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/veterinaria , Activación Enzimática , Glioma/genética , Glioma/patología , Glioma/veterinaria , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Neuroblastoma/genética , Neuroblastoma/patología , Neuroblastoma/veterinaria , Neuronas , Proteínas Tirosina Quinasas/genética , Ratas , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Reactive oxygen species (ROS) are implicated in reperfusion injury after transient focal cerebral ischemia. The antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD), is one of the major means by which cells counteract the deleterious effects of ROS after ischemia. Recently, we reported that when Tat-SOD fusion protein is transduced into pancreatic beta cells it protects the beta cells from destruction by relieving oxidative stress in ROS-implicated diabetes (Eum et al., 2004). In the present study, we investigated the protective effects of Tat-SOD fusion protein against neuronal cell death and ischemic insults. When Tat-SOD was added to the culture medium of neuronal cells, it rapidly entered the cells and protected them against paraquat-induced cell death. Immunohistochemical analysis revealed that Tat-SOD injected intraperitoneally (i.p.) into mice has access to various tissues including brain neurons. When i.p. injected into gerbils, Tat-SOD prevented neuronal cell death in the hippocampus in response to transient fore-brain ischemia. These results suggest that Tat-SOD provides a strategy for therapeutic delivery in various hu-man diseases, including stroke, related to this anti-oxidant enzyme or to ROS.
Asunto(s)
Astrocitos/citología , Astrocitos/efectos de los fármacos , Ataque Isquémico Transitorio/tratamiento farmacológico , Daño por Reperfusión/prevención & control , Superóxido Dismutasa/uso terapéutico , Animales , Antioxidantes/uso terapéutico , Muerte Celular/efectos de los fármacos , Productos del Gen tat/uso terapéutico , Gerbillinae , Masculino , Ratones , Ratones Endogámicos ICR , Estrés Oxidativo/fisiología , Proteínas Recombinantes de Fusión/uso terapéutico , Transducción GenéticaRESUMEN
Poor membrane permeability of proteins is a major limitation of protein therapy. In a previous study, we showed that the minimal sequence required for efficient transduction of Tat-GFP is the basic domain from 49-57 of HIV-1 Tat called the protein transduction domain (PTD. Here we have generated HIV-1 Tat PTD GFP fusion proteins in which HIV-1 Tat PTD is fused with the N- and/or C-termini of GFP. The various GFP fusion proteins were purified from Escherichia coli and characterized for their ability to enter mammalian cells using Western blot analysis, confocal microscopy and flow cytometry. The GFP fusion protein with Tat PTD at its C-terminus was taken up as efficiently as the GFP fusion protein with Tat PTD at its N-terminus. However, the same protein with PTDs at its both termini was taken up even more efficiently. All the GFP fusion proteins were present in both the nucleus and cytosol of the transduced cells. Uptake was lower at 4 degrees C than at 37 degrees C. The availability of the expression vectors developed in this study may help to devise novel strategies in the rational development of protein-based drugs.
Asunto(s)
Productos del Gen tat/metabolismo , VIH-1/metabolismo , Genes Reporteros , Células HeLa , Humanos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Temperatura , Transducción Genética , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
We previously reported that Tat-Cu,Zn-superoxide dismutase (Tat-SOD), a major antioxidant enzyme, can be directly transduced into mammalian cells and skin [Kwon et al. (2000); Park et al. (2002)]. To enhance the therapeutic potential of Tat-SOD in the treatment of various disorders, we screened a number of natural products for their ability to increase transduction efficiency. Ginsenosides were effective with cultured HeLa cells and enhanced the penetration of Tat-SOD into both the epidermis and the dermis of the subcutaneous layer when sprayed on mice skin. Although their mechanism of action is not fully understood we believe that ginsenosides may be useful cofactors with this antioxidant enzyme in anti-aging cosmetics or as a therapeutic protein in disorders related to reactive-oxygen species.
Asunto(s)
Productos del Gen tat/metabolismo , Ginsenósidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Superóxido Dismutasa/metabolismo , Animales , Productos del Gen tat/genética , Células HeLa , Humanos , Inmunohistoquímica , Ratones , Transporte de Proteínas/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Piel/metabolismo , Superóxido Dismutasa/genéticaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD), which contains a high proportion of arginine and lysine residues, is responsible for highly efficient protein transduction through the plasma membrane. To identify the role of the PTD sequence motif in transduction, various deletions and substitutions were introduced into the PTD. Tat-green fluorescent protein (GFP) fusion proteins, containing various lengths of the Tat PTD, were expressed and the extent of their transduction into mammalian cells was analysed by Western blot analysis and fluorescence microscopy. Deletion analysis of PTD mapped to a nine amino acid motif (residues 49-57: RKKRRQRRR) sufficient for transduction. Further deletion of this Tat basic domain either at the N terminus or at the C terminus significantly decreased transduction efficiency. The transduction efficiencies of GFPs fused to nine consecutive lysine (9Lys-GFP) or arginine (9Arg-GFP) residues were similar to that of Tat(49-57)-GFP. The transduced proteins localized to both the nucleus and the cytosol, as assessed by confocal microscopy and Western blot analysis of subcellular fractions from transduced cells. Thus, the availability of recombinant GFP fusion proteins facilitates the simple and specific identification of protein transduction mediated by these peptide sequences. The modified PTD sequences designed in this study may provide useful tools necessary for delivering therapeutic proteins/peptides into cells.