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2P-0461 SREBPs are negatively regulated by SUMO-1 modification
Tuesday September 30, 2003: Poster Session Adhesion molecules performed by the subcutaneous injection of Humalin N (4units). After 3 day of STZ injection, PH was performed. And 1days or 7 days after PH, liver was removed and used for mRNA measurement. Results: It was revealed that, on the first day after PH, expression of hepatic SREBP-1 mRNA decreased, although FAS activity was increased. STZ induced diabetic rat showed a significant decrease of SREBP-1 mRNA, but the regeneration of hepatocytes were observed as usual. These data indicate that the decrease of SREBP-1 is not be the initiation signal of regeneration of hepatocytes. When STZ induced diabetic rats were treated with insulin, SREBP-1 mRNA increased and hepatic regeneration was occurred more than that of untreated diabetic rats, significantly. This data indicated that SREBP-1 might be the enhancer of hepatic regeneration. Conclusion: SREBP-1 plays not as the initiator but as the enhancer role in the hepatocyte regeneration after partial hepatectomy. 2P-0461
SREBPs are negatively regulated by SUMO-1 modification
R. Sato 1 , Y. Hirano 2 , S. Murata 3 , K. Tanaka 3 , M. Shimizu 2 . 1 Department of Applied Biological Chemistry, University of Tokyo; 2 University of Tokyo; 3 Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
2P-0462
Impact of adipophilin expression on lipid accumulation in human macrophages: Potential role in atherogenesis
G. Larigauderie, C. Furman, C. Lasselin, C. Copin, J.-C. Fruchart, G. Castro, M. Rouis. INSERM U545, Lille Pasteur Institute, Lille, France Macrophage-derived foam cells, which occur in abundance in most stages of the atherosclerotic lesion, play a major role in atheroma plaque rupture. Lipid loading has been reported to modulate expression of several genes such as adipophilin, a protein with a putative role in lipid handling. We have previously shown that adipophilin expression in atherosclerotic lesions was more than 3 fold higher than its expression in healthy areas of the same arteries. In this study, we have investigated the role of adipophilin expression in lipid accumulation in THP-1-derived macrophages. Thus, THP-1 macrophages overexpressing human adipophilin were incubated 48h with acetylated LDL (acLDL). Our data showed 76% and 45% increase of triglycerides (TG) and total cholesterol (TC) respectively in adipophilin transfected cells versus control cells. In addition, following 24h incubation of the lipid loaded cells with apoA-I, cellular cholesterol efflux was significantly reduced from the adipophilin transfected cells by 47% when compared to control cells. Moreover, the impact of adipophilin gene silencing on lipid accumulation and cholesterol efflux, using a specific siRNA directed against adipophilin mRNA, showed a 50% and 44% reduction of TG and TC respectively as well as a 50% decrease in cholesterol efflux. In conclusion, both overexpression and down-regulation of adipophilin reduced cholesterol efflux suggesting a role for adipophilin in the regulation or localisation of the cholesterol pools available for the ABCA1 pathway. Moreover, adipophilin overexpression promoted TC and TG accumulation, whereas the suppression of adipophilin expression reduced both TC and TG content in human THP-1 macrophages. Our results suggested that stimulation of adipophilin expression in macrophages contribute to lipid storage which might lead to plaque rupture. Therefore, adipophilin might constitute a new target gene in atherosclerosis treatment.
Regulation of the LDL receptor gene expression by bile acids
M. Nakahara 1 , Y. Yokoi 1 , H. Fujii 2 , M. Shimizu 1 , R. Sato 1 . 1 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo; 2 Graduate School of Medical and Dental Science, Niigata University, Japan Recent studies have indicated that bile acids regulate the expression of several genes involved in bile acid and lipid metabolism as ligands for farnesoid X receptor (FXR). We here report that bile acids are directly able to govern cholesterol metabolism by a novel mechanism. We show that chenodeoxycholic acid (CDCA) enhances low density lipoprotein (LDL) receptor gene expression in HepG2 cells. The proteolytic activation of sterol regulatory element-binding protein-2 (SREBP-2), a major regulator for LDL receptor gene expression, is not affected by CDCA. Both deoxycholic acid (DCA) and lithocholic acid (LCA) as well as CDCA, but not ursodeoxycholic acid (UDCA), increase the mRNA level for the LDL receptor, even when Hep G2 cells are cultured with 25-hydroxycholesterol, a potent suppressor of gene expression for the LDL receptor. Although it seems possible that FXR might be involved in genetic regulation, both reporter assays with a reporter gene containing the LDL receptor promoter, as well as northern blot analysis reveal that FXR has exerts no genetic effects. On the other hand, inhibition of mitogen-activated protein (MAP) kinase activities, which are found to be induced by CDCA, abolishes the CDCA-mediated up-regulation of LDL receptor gene expression. We further demonstrate that CDCA stabilizes LDL receptor mRNA, and that the MAP kinase inhibitors accelerate its turnover. And we show that this stabilization is mediated by the 3’ untranslated region of the LDL receptor. Taken together, these results indicate that bile acids increase LDL uptake and the intracellular cholesterol levels through the activation of MAP kinase cascades in conjunction with a down-regulation of bile acid biosynthesis by FXR.
ADHESION MOLECULES 2P-0464
Carvedilol, a beta-adrenoceptor antagonist with antioxidant activity, attenuates TNF-alpha stimulated expression of VCAM-1 and E-selectin, and adhesiveness to mononuclear cells in human aortic endothelial cells
J.-W. Chen, F.-Y. Lin, Y.-H. Chen, T.-L. Yang, Y.-L. Chen, S.-J. Lin. National Yang-Ming University School of Medicine, Taiwan Background: The beneficial effects of carvedilol, a beta-adrenoceptor antagonist, in decreasing cardiovascular mortality might be attributed to its antioxidant properties. Since oxidative stress may play an important role in atherogenesis. We then examined the direct effects of carvedilol on endothelial-adhesivenss to human mononuclear cells (MNCs), an in vitro sign of atherogenesis, and the expression of adhesion molecules in human aortic endothelial cells (HAECs) stimulated by tumor necrosis factor-alpha (TNF-α). Methods and Results: Circulating MNCs were isolated from the peripheral blood of healthy subjects. Compared to control condition, pretreatment of carvedilol (10 mM for 18 hours) or probucol (5 mM for 18 hours) but not propanolol significantly decreased TNF-α-stimulated adhesiveness of cultured HAECs to MNCs (51.1±4.0% and 52.1±6.2% inhibition, p<0.05, respectively). Cell ELISA study showed that pretreatment with carvedilol significantly inhibited TNF-α-stimulated VCAM-1 and E-selectin (66.0±2.0% and 55.6±1.0% of control, p<0.05, respectively) but not ICAM-1 expression whereas pretreatment with probucol inhibited only VCAM-1 expression (79.0±5.0% of control, p<0.05) in HAECs. Propranolol did not alter the expression of any adhesion molecules. The above findings were also confirmed by Western blotting study. Further, carvedilol significantly inhibited TNF-α-stimulated intracellular reactive oxygen species (ROS) production and the activation of redox sensitive transcription pathways including both nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) in HAECs. Conclusions: Carvedilol may directly prevent in vitro atherogenesis by inhibiting intracellular ROS production, NF-kB and AP-1 activation, and VCAM-1 as well as E-selectin expression in TNF-α-stimulated HAECs, suggesting the potential implication of carvedilol to clinical atherosclerosis disease.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
TUESDAY
Sterol regulatory element-binding proteins (SREBPs) are major transcription factors that activate the genes involved in cholesterol and fatty acid biosynthesis. We here report that the nuclear forms of SREBPs are modified by the small ubiquitin-related modifier (SUMO)-1. Mutational analyses identified two major sumoylation sites (Lys123 and Lys418) in SREBP-1a and a single site (Lys464) in SREBP-2. Mutant SREBPs lacking one or two sumoylation sites exhibited increased transactivation capacity on an SREBP-responsive promoter. Overexpression of SUMO-1 reduced while its dominant negative form increased mRNA levels of SREBP-responsive genes. Nuclear SREBPs interacted with the SUMO-1-conjugating enzyme Ubc9, and overexpression of a dominant negative form of Ubc9 increased the mRNA levels of SREBPresponsive genes. Pulse-chase experiments revealed that sumoylation did not affect the degradation of SREBPs through the ubiquitin-proteasome pathway. In vitro ubiquitylation assay showed no competition between ubiquitin and SUMO-1 for the same lysine. Considered together, our results indicate that SUMO-1 modification suppresses the transactivation capacity of nuclear SREBPs in a manner different from the negative regulatory mechanism mediated by proteolysis.
2P-0463 ∗
141
SREBPs are negatively regulated by SUMO-1 modification
R. Sato 1 , Y. Hirano 2 , S. Murata 3 , K. Tanaka 3 , M. Shimizu 2 . 1 Department of Applied Biological Chemistry, University of Tokyo; 2 University of Tokyo; 3 Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
2P-0462
Impact of adipophilin expression on lipid accumulation in human macrophages: Potential role in atherogenesis
G. Larigauderie, C. Furman, C. Lasselin, C. Copin, J.-C. Fruchart, G. Castro, M. Rouis. INSERM U545, Lille Pasteur Institute, Lille, France Macrophage-derived foam cells, which occur in abundance in most stages of the atherosclerotic lesion, play a major role in atheroma plaque rupture. Lipid loading has been reported to modulate expression of several genes such as adipophilin, a protein with a putative role in lipid handling. We have previously shown that adipophilin expression in atherosclerotic lesions was more than 3 fold higher than its expression in healthy areas of the same arteries. In this study, we have investigated the role of adipophilin expression in lipid accumulation in THP-1-derived macrophages. Thus, THP-1 macrophages overexpressing human adipophilin were incubated 48h with acetylated LDL (acLDL). Our data showed 76% and 45% increase of triglycerides (TG) and total cholesterol (TC) respectively in adipophilin transfected cells versus control cells. In addition, following 24h incubation of the lipid loaded cells with apoA-I, cellular cholesterol efflux was significantly reduced from the adipophilin transfected cells by 47% when compared to control cells. Moreover, the impact of adipophilin gene silencing on lipid accumulation and cholesterol efflux, using a specific siRNA directed against adipophilin mRNA, showed a 50% and 44% reduction of TG and TC respectively as well as a 50% decrease in cholesterol efflux. In conclusion, both overexpression and down-regulation of adipophilin reduced cholesterol efflux suggesting a role for adipophilin in the regulation or localisation of the cholesterol pools available for the ABCA1 pathway. Moreover, adipophilin overexpression promoted TC and TG accumulation, whereas the suppression of adipophilin expression reduced both TC and TG content in human THP-1 macrophages. Our results suggested that stimulation of adipophilin expression in macrophages contribute to lipid storage which might lead to plaque rupture. Therefore, adipophilin might constitute a new target gene in atherosclerosis treatment.
Regulation of the LDL receptor gene expression by bile acids
M. Nakahara 1 , Y. Yokoi 1 , H. Fujii 2 , M. Shimizu 1 , R. Sato 1 . 1 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo; 2 Graduate School of Medical and Dental Science, Niigata University, Japan Recent studies have indicated that bile acids regulate the expression of several genes involved in bile acid and lipid metabolism as ligands for farnesoid X receptor (FXR). We here report that bile acids are directly able to govern cholesterol metabolism by a novel mechanism. We show that chenodeoxycholic acid (CDCA) enhances low density lipoprotein (LDL) receptor gene expression in HepG2 cells. The proteolytic activation of sterol regulatory element-binding protein-2 (SREBP-2), a major regulator for LDL receptor gene expression, is not affected by CDCA. Both deoxycholic acid (DCA) and lithocholic acid (LCA) as well as CDCA, but not ursodeoxycholic acid (UDCA), increase the mRNA level for the LDL receptor, even when Hep G2 cells are cultured with 25-hydroxycholesterol, a potent suppressor of gene expression for the LDL receptor. Although it seems possible that FXR might be involved in genetic regulation, both reporter assays with a reporter gene containing the LDL receptor promoter, as well as northern blot analysis reveal that FXR has exerts no genetic effects. On the other hand, inhibition of mitogen-activated protein (MAP) kinase activities, which are found to be induced by CDCA, abolishes the CDCA-mediated up-regulation of LDL receptor gene expression. We further demonstrate that CDCA stabilizes LDL receptor mRNA, and that the MAP kinase inhibitors accelerate its turnover. And we show that this stabilization is mediated by the 3’ untranslated region of the LDL receptor. Taken together, these results indicate that bile acids increase LDL uptake and the intracellular cholesterol levels through the activation of MAP kinase cascades in conjunction with a down-regulation of bile acid biosynthesis by FXR.
ADHESION MOLECULES 2P-0464
Carvedilol, a beta-adrenoceptor antagonist with antioxidant activity, attenuates TNF-alpha stimulated expression of VCAM-1 and E-selectin, and adhesiveness to mononuclear cells in human aortic endothelial cells
J.-W. Chen, F.-Y. Lin, Y.-H. Chen, T.-L. Yang, Y.-L. Chen, S.-J. Lin. National Yang-Ming University School of Medicine, Taiwan Background: The beneficial effects of carvedilol, a beta-adrenoceptor antagonist, in decreasing cardiovascular mortality might be attributed to its antioxidant properties. Since oxidative stress may play an important role in atherogenesis. We then examined the direct effects of carvedilol on endothelial-adhesivenss to human mononuclear cells (MNCs), an in vitro sign of atherogenesis, and the expression of adhesion molecules in human aortic endothelial cells (HAECs) stimulated by tumor necrosis factor-alpha (TNF-α). Methods and Results: Circulating MNCs were isolated from the peripheral blood of healthy subjects. Compared to control condition, pretreatment of carvedilol (10 mM for 18 hours) or probucol (5 mM for 18 hours) but not propanolol significantly decreased TNF-α-stimulated adhesiveness of cultured HAECs to MNCs (51.1±4.0% and 52.1±6.2% inhibition, p<0.05, respectively). Cell ELISA study showed that pretreatment with carvedilol significantly inhibited TNF-α-stimulated VCAM-1 and E-selectin (66.0±2.0% and 55.6±1.0% of control, p<0.05, respectively) but not ICAM-1 expression whereas pretreatment with probucol inhibited only VCAM-1 expression (79.0±5.0% of control, p<0.05) in HAECs. Propranolol did not alter the expression of any adhesion molecules. The above findings were also confirmed by Western blotting study. Further, carvedilol significantly inhibited TNF-α-stimulated intracellular reactive oxygen species (ROS) production and the activation of redox sensitive transcription pathways including both nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) in HAECs. Conclusions: Carvedilol may directly prevent in vitro atherogenesis by inhibiting intracellular ROS production, NF-kB and AP-1 activation, and VCAM-1 as well as E-selectin expression in TNF-α-stimulated HAECs, suggesting the potential implication of carvedilol to clinical atherosclerosis disease.
XIIIth International Symposium on Atherosclerosis, September 28–October 2, 2003, Kyoto, Japan
TUESDAY
Sterol regulatory element-binding proteins (SREBPs) are major transcription factors that activate the genes involved in cholesterol and fatty acid biosynthesis. We here report that the nuclear forms of SREBPs are modified by the small ubiquitin-related modifier (SUMO)-1. Mutational analyses identified two major sumoylation sites (Lys123 and Lys418) in SREBP-1a and a single site (Lys464) in SREBP-2. Mutant SREBPs lacking one or two sumoylation sites exhibited increased transactivation capacity on an SREBP-responsive promoter. Overexpression of SUMO-1 reduced while its dominant negative form increased mRNA levels of SREBP-responsive genes. Nuclear SREBPs interacted with the SUMO-1-conjugating enzyme Ubc9, and overexpression of a dominant negative form of Ubc9 increased the mRNA levels of SREBPresponsive genes. Pulse-chase experiments revealed that sumoylation did not affect the degradation of SREBPs through the ubiquitin-proteasome pathway. In vitro ubiquitylation assay showed no competition between ubiquitin and SUMO-1 for the same lysine. Considered together, our results indicate that SUMO-1 modification suppresses the transactivation capacity of nuclear SREBPs in a manner different from the negative regulatory mechanism mediated by proteolysis.
2P-0463 ∗
141