Jamila Najib

Activation of the peroxisome proliferator-activated receptor gamma promotes the development of colon tumors in C57BL/6J-APCMin/+ mice.

The development of colorectal cancer, one of the most frequent cancers, is influenced by prostaglandins and fatty acids. Decreased prostaglandin production, seen in mice with mutations in the cyclooxygenase 2 gene or in animals and humans treated with cyclooxygenase inhibitors, prevents or attenuates colon cancer development. There is also a strong correlation between the intake of fatty acids from animal origin and colon cancer. Therefore, the peroxisome proliferator-activated receptor γ (PPARγ; ref. 7), a downstream transcriptional mediator for prostaglandins and fatty acids which is highly expressed in the colon, may be involved in this process. Activation of PPARγ by two different synthetic agonists increased the frequency and size of colon tumors in C57BL/6J-APCMin /+ mice, an animal model susceptible to intestinal neoplasia. Tumor frequency was only increased in the colon, and did not change in the small intestine, coinciding with the colon-restricted expression of PPARγ. Treatment with PPARγ agonists increased β-catenin levels both in the colon of C57BL/6J-APCMin/+ mice and in HT-29 colon carcinoma cells. Genetic abnormalities in the Wnt/wingless/APC pathway, which enhance the transcriptional activity of the β-catenin-T-cell factor/lymphoid enhancer factor 1 transcription complex, often underly the development of colon tumors. Our data indicate that PPARγ activation modifies the development of colon tumors in C57BL/6J-APCMin/+ mice.

Peroxisome Proliferator-Activated Receptor Activators Inhibit Thrombin-Induced Endothelin-1 Production in Human Vascular Endothelial Cells by Inhibiting the Activator Protein-1 Signaling Pathway

Endothelin-1 (ET-1), a 21-amino acid vasoactive peptide mainly produced by vascular endothelial cells, is involved in the regulation of vascular tone and smooth muscle cell proliferation. Peroxisome proliferator-activated receptors (PPARs), key players in lipid and glucose metabolism, have been implicated in metabolic disorders that are predisposing to atherosclerosis. Because of the potential role of ET-1 in vascular disorders such as hypertension and atherosclerosis, we investigated the regulation of ET-1 expression by PPAR activators. Western blot and reverse transcription-polymerase chain reaction analyses demonstrated that both PPARalpha and PPARgamma are expressed in human coronary artery endothelial cells as well as in endothelial cell lines such as HMEC-1 and ECV304. In bovine aortic endothelial cells and HMEC-1 cells, both PPARalpha and PPARgamma ligands inhibited thrombin-induced ET-1 secretion, whereas basal ET-1 secretion was only slightly suppressed. Reverse transcription-polymerase chain reaction experiments showed that this inhibition of ET-1 production occurs at the gene expression level. Using transient transfection assays, we demonstrated that PPARs downregulate thrombin-activated transcription of the human ET-1 promoter. Transactivation studies with c-Jun and c-Fos expression plasmids indicated that PPARs negatively interfere with the activator protein-1 signaling pathway, which mediates thrombin activation of ET-1 gene transcription. Furthermore, electrophoretic mobility shift assays demonstrated that PPAR activators reduce the thrombin-stimulated binding activity of bovine aortic endothelial cell nuclear extracts as well as c-Jun binding to an activator protein-1 consensus site. Taken together, these data indicate that (1) both PPARalpha and PPARgamma are expressed in human vascular endothelial cells and (2) PPAR activators inhibit thrombin-induced ET-1 biosynthesis, indicating a novel role for PPARs in vascular endothelial function.

Regulation of Peroxisome Proliferator-Activated Receptor γ Expression by Adipocyte Differentiation and Determination Factor 1/Sterol Regulatory Element Binding Protein 1: Implications for Adipocyte Differentiation and Metabolism

ABSTRACT Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor implicated in adipocyte differentiation and insulin sensitivity. We investigated whether PPARγ expression is dependent on the activity of adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1 (ADD-1/SREBP-1), another transcription factor associated with both adipocyte differentiation and cholesterol homeostasis. Ectopic expression of ADD-1/SREBP-1 in 3T3-L1 and HepG2 cells induced endogenous PPARγ mRNA levels. The related transcription factor SREBP-2 likewise induced PPARγ expression. In addition, cholesterol depletion, a condition known to result in proteolytic activation of transcription factors of the SREBP family, induced PPARγ expression and improved PPRE-driven transcription. The effect of the SREBPs on PPARγ expression was mediated through the PPARγ1 and -3 promoters. Both promoters contain a consensus E-box motif that mediates the regulation of the PPARγ gene by ADD-1/SREBP-1 and SREBP-2. These results suggest that PPARγ expression can be controlled by the SREBP family of transcription factors and demonstrate new interactions between transcription factors that can regulate different pathways of lipid metabolism.

Developmental and pharmacological regulation of apolipoprotein C-II gene expression. Comparison with apo C-I and apo C-III gene regulation.

Increased plasma triglyceride concentrations are often observed in metabolic disorders predisposing to coronary heart disease. Among the major determinants of plasma triglyceride metabolism are the apolipoproteins (apos) of the C class, C-I, C-II, and C-III. Whereas physiological concentrations of apo C-II are required for lipolysis of triglycerides by lipoprotein lipase (LPL), overexpression of all 3 C apolipoproteins leads to hypertriglyceridemia. In the present study, we investigated apo C-II gene regulation under conditions associated with profound changes in plasma triglyceride metabolism, ie, during postnatal development and after treatment with the triglyceride-lowering fibrate drugs, and compared its expression to that of apo C-I and apo C-III. Whereas the expression of both apo C-I and apo C-III is low in fetal liver, increases gradually after birth, and attains maximal levels after weaning, apo C-II gene expression is already detectable in the fetal liver, increases rapidly immediately after birth, and remains elevated throughout suckling. Thus, the increased ingestion of lipids during suckling is met by an earlier induction of apo C-II, the obligatory activator for LPL, compared with apo C-III and apo C-I, which antagonize triglyceride catabolism. Treatment of rats with fibrates decreased apo C-II gene expression in the liver, but not in the intestine, whereas apo C-I gene expression did not change. The decrease of liver apo C-II mRNA levels after fenofibrate occurred in a time- and dose-dependent manner and was reversible but appeared less pronounced than the decrease of apo C-III mRNA. Apo C-II mRNA levels were not affected after treatment with BRL49653, a peroxisome proliferator-activated receptor (PPAR)gamma-specific ligand, suggesting that fibrates act on apo C-II expression via PPARalpha. Addition of fenofibric acid to primary rat and human hepatocytes resulted in a decrease of apo C-II expression. In conclusion, fibrates decrease gene expression of apo C-II and apo C-III, but not apo C-I, in rat and human hepatocytes. This decrease of apo C-II and apo C-III gene expression, together with a lowered apo C-III to apo C-II ratio, should result in an improved clearance of triglyceride-rich remnant lipoproteins from plasma, without hampering triglyceride lipolysis by LPL.

Expression of Human Apolipoprotein A-I/C-III/A-IV Gene Cluster in Mice Induces Hyperlipidemia but Reduces Atherogenesis

The apolipoprotein (apo)A-I/C-III/A-IV gene cluster is involved in lipid metabolism and atherosclerosis. Overexpression of apoC-III in mice causes hypertriglyceridemia and induces atherogenesis, whereas overexpression of apoA-I or apoA-IV increases cholesterol in plasma high density lipoprotein (HDL) and protects against atherosclerosis. Each gene has been studied alone in transgenic mice but not in combination as the entire cluster. To determine which phenotype is produced by the expression of the entire gene cluster, transgenic mice were generated with a 33-kb human DNA fragment. The results showed that the transgene contained the necessary elements to direct hepatic and intestinal expression of the 3 genes. In the pooled data, plasma concentrations were 257±9, 7.1±0.5, and 1.0±0.2 mg/dL for human apoA-I, apoC-III, and apoA-IV, respectively (mean±SEM). Concentrations of these apolipoproteins were higher in males than in females. Human apoA-I and apoC-III concentrations were positively correlated, suggesting that they are coregulated. Transgenic mice exhibited gross hypertriglyceridemia and accumulation of apoB48–containing triglyceride-rich lipoproteins. Plasma triglyceride and cholesterol concentrations were correlated positively with human apoC-III concentration, and HDL cholesterol was correlated with apoA-I concentration. In an apoE-deficient background, despite being markedly hypertriglyceridemic, cluster transgenic animals compared with nontransgenic animals showed a 61% reduction in atherosclerosis. This suggests that apoA-I and/or apoA-IV can protect against atherosclerosis even in the presence of severe hyperlipidemia. These mice provide a new model for studies of the regulation of the 3 human genes in combination.

Formation of Nε-Dnp lysine during deprotection of Nε-Fmoc lysine in Nim-Dnp-histidine-containing peptides

Abstract The 2,4-dinitrophenyl group used for histidine side chain protection is not stable during aminolytic removal of Fmoc protecting groups. In the presence