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1.  Insulin-Mediated Down-Regulation of Apolipoprotein A5 Gene Expression through the Phosphatidylinositol 3-Kinase Pathway: Role of Upstream Stimulatory Factor 
Molecular and Cellular Biology  2005;25(4):1537-1548.
The apolipoprotein A5 gene (APOA5) has been repeatedly implicated in lowering plasma triglyceride levels. Since several studies have demonstrated that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 is regulated by insulin. Here, we show that cell lines and mice treated with insulin down-regulate APOA5 expression in a dose-dependent manner. Furthermore, we found that insulin decreases human APOA5 promoter activity, and subsequent deletion and mutation analyses uncovered a functional E box in the promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that this APOA5 E box binds upstream stimulatory factors (USFs). Moreover, in transfection studies, USF1 stimulates APOA5 promoter activity, and the treatment with insulin reduced the binding of USF1/USF2 to the APOA5 promoter. The inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway abolished insulin's effect on APOA5 gene expression, while the inhibition of the P70 S6 kinase pathway with rapamycin reversed its effect and increased APOA5 gene expression. Using an oligonucleotide precipitation assay for USF from nuclear extracts, we demonstrate that phosphorylated USF1 fails to bind to the APOA5 promoter. Taken together, these data indicate that insulin-mediated APOA5 gene transrepression could involve a phosphorylation of USFs through the PI3K and P70 S6 kinase pathways that modulate their binding to the APOA5 E box and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in men showed a decrease in the plasma ApoAV level. These results suggest a potential contribution of the APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.
doi:10.1128/MCB.25.4.1537-1548.2005
PMCID: PMC548024  PMID: 15684402
2.  Gene-Gene Interaction between APOA5 and USF1: Two Candidate Genes for the Metabolic Syndrome 
Obesity Facts  2009;2(4):235-242.
Summary
Objective
The metabolic syndrome, a major cluster of risk factors for cardiovascular diseases, shows increasing prevalence worldwide. Several studies have established associations of both apolipoprotein A5 (APOA5) gene variants and upstream stimulatory factor 1 (USF1) gene variants with blood lipid levels and metabolic syndrome. USF1 is a transcription factor for APOA5.
Methods
We investigated a possible interaction between these two genes on the risk for the metabolic syndrome, using data from the German population-based KORA survey 4 (1,622 men and women aged 55–74 years). Seven APOA5 single nucleotide polymorphisms (SNPs) were analyzed in combination with six USF1 SNPs, applying logistic regression in an additive model adjusting for age and sex and the definition for metabolic syndrome from the National Cholesterol Education Program's Adult Treatment Panel III (NCEP (AIII)) including medication.
Results
The overall prevalence for metabolic syndrome was 41%. Two SNP combinations showed a nominal gene-gene interaction (p values 0.024 and 0.047). The effect of one SNP was modified by the other SNP, with a lower risk for the metabolic syndrome with odds ratios (ORs) between 0.33 (95% CI = 0.13–0.83) and 0.40 (95% CI = 0.15–1.12) when the other SNP was homozygous for the minor allele. Nevertheless, none of the associations remained significant after correction for multiple testing.
Conclusion
Thus, there is an indication of an interaction between APOA5 and USF1 on the risk for metabolic syndrome.
doi:10.1159/000227288
PMCID: PMC2919429  PMID: 20054229
Metabolic syndrome; Cardiovascular risk; SNP; APOA5; USF1
3.  Risk Alleles of USF1 Gene Predict Cardiovascular Disease of Women in Two Prospective Studies 
PLoS Genetics  2006;2(5):e69.
Upstream transcription factor 1 (USF1) is a ubiquitously expressed transcription factor controlling several critical genes in lipid and glucose metabolism. Of some 40 genes regulated by USF1, several are involved in the molecular pathogenesis of cardiovascular disease (CVD). Although the USF1 gene has been shown to have a critical role in the etiology of familial combined hyperlipidemia, which predisposes to early CVD, the gene's potential role as a risk factor for CVD events at the population level has not been established. Here we report the results from a prospective genetic–epidemiological study of the association between the USF1 variants, CVD, and mortality in two large Finnish cohorts. Haplotype-tagging single nucleotide polymorphisms exposing all common allelic variants of USF1 were genotyped in a prospective case-cohort design with two distinct cohorts followed up during 1992–2001 and 1997–2003. The total number of follow-up years was 112,435 in 14,140 individuals, of which 2,225 were selected for genotyping based on the case-cohort study strategy. After adjustment for conventional risk factors, we observed an association of USF1 with CVD and mortality among females. In combined analysis of the two cohorts, female carriers of a USF1 risk haplotype had a 2-fold risk of a CVD event (hazard ratio [HR] 2.02; 95% confidence interval [CI] 1.16–3.53; p = 0.01) and an increased risk of all-cause mortality (HR 2.52; 95% CI 1.46–4.35; p = 0.0009). A putative protective haplotype of USF1 was also identified. Our study shows how a gene identified in exceptional families proves to be important also at the population level, implying that allelic variants of USF1 significantly influence the prospective risk of CVD and even all-cause mortality in females.
Synopsis
Better characterization of molecular events resulting in cardiovascular disease (CVD) requires elucidation of genetic background of CVD. After a CVD candidate gene is identified in family-based studies or case-control studies, population-based prospective studies are needed to demonstrate any potential impact of allelic variants on the CVD risk at the population level. This study addresses the role of different alleles of the upstream transcription factor 1 (USF1) gene, encoding a transcription factor and originally associated with familial combined hyperlipidemia in rare families with multiple affected individuals. The product of USF1 regulates numerous genes of lipid and glucose metabolism, and the authors show in large population cohorts that specific alleles of USF1 are associated with the risk of CVD and all-cause mortality among females. The study implies an interesting female-specific risk effect, and should stimulate additional studies of the sex-specific CVD risk genes in different populations.
doi:10.1371/journal.pgen.0020069
PMCID: PMC1458962  PMID: 16699592
4.  Risk Alleles of USF1 Gene Predict Cardiovascular Disease of Women in Two Prospective Studies 
PLoS Genetics  2006;2(5):e69.
Upstream transcription factor 1 (USF1) is a ubiquitously expressed transcription factor controlling several critical genes in lipid and glucose metabolism. Of some 40 genes regulated by USF1, several are involved in the molecular pathogenesis of cardiovascular disease (CVD). Although the USF1 gene has been shown to have a critical role in the etiology of familial combined hyperlipidemia, which predisposes to early CVD, the gene's potential role as a risk factor for CVD events at the population level has not been established. Here we report the results from a prospective genetic–epidemiological study of the association between the USF1 variants, CVD, and mortality in two large Finnish cohorts. Haplotype-tagging single nucleotide polymorphisms exposing all common allelic variants of USF1 were genotyped in a prospective case-cohort design with two distinct cohorts followed up during 1992–2001 and 1997–2003. The total number of follow-up years was 112,435 in 14,140 individuals, of which 2,225 were selected for genotyping based on the case-cohort study strategy. After adjustment for conventional risk factors, we observed an association of USF1 with CVD and mortality among females. In combined analysis of the two cohorts, female carriers of a USF1 risk haplotype had a 2-fold risk of a CVD event (hazard ratio [HR] 2.02; 95% confidence interval [CI] 1.16–3.53; p = 0.01) and an increased risk of all-cause mortality (HR 2.52; 95% CI 1.46–4.35; p = 0.0009). A putative protective haplotype of USF1 was also identified. Our study shows how a gene identified in exceptional families proves to be important also at the population level, implying that allelic variants of USF1 significantly influence the prospective risk of CVD and even all-cause mortality in females.
Synopsis
Better characterization of molecular events resulting in cardiovascular disease (CVD) requires elucidation of genetic background of CVD. After a CVD candidate gene is identified in family-based studies or case-control studies, population-based prospective studies are needed to demonstrate any potential impact of allelic variants on the CVD risk at the population level. This study addresses the role of different alleles of the upstream transcription factor 1 (USF1) gene, encoding a transcription factor and originally associated with familial combined hyperlipidemia in rare families with multiple affected individuals. The product of USF1 regulates numerous genes of lipid and glucose metabolism, and the authors show in large population cohorts that specific alleles of USF1 are associated with the risk of CVD and all-cause mortality among females. The study implies an interesting female-specific risk effect, and should stimulate additional studies of the sex-specific CVD risk genes in different populations.
doi:10.1371/journal.pgen.0020069
PMCID: PMC1458962  PMID: 16699592
5.  Association Analysis of Allelic Variants of USF1 in Coronary Atherosclerosis 
Objective
USF1 regulates the transcription of more than 40 cardiovascular related genes and is well established as a gene associated with familial combined hyperlipidemia, a condition increasing the risk for coronary heart disease. No detailed data, however, exists on the impact of this gene to the critical outcome at the tissue level: different types of atherosclerotic lesions.
Methods and Results
We analyzed the USF1 in 2 autopsy series of altogether 700 middle-aged men (the Helsinki Sudden Death Study) with quantitative morphometric measurements of coronary atherosclerosis. SNP rs2516839, tagging common USF1 haplotypes, associated with the presence of several types of atherosclerotic lesions, particularly with the proportion of advanced atherosclerotic plaques (P=0.02) and area of calcified lesions (P<0.001) of the coronary arteries. Importantly, carriers of risk alleles of rs2516839 also showed a 2-fold risk for sudden cardiac death (genotype TT versus CC; OR 2.10, 95% CI 1.17 to 3.75, P=0.04). The risk effect of rs2516839 was present also in aorta samples of the men.
Conclusions
Our findings in this unique study sample suggest that USF1 contributes to atherosclerosis, the pathological arterial wall phenotype resulting in coronary heart disease and in its most dramatic consequence—sudden cardiac death.
doi:10.1161/ATVBAHA.107.156463
PMCID: PMC2687549  PMID: 18276913
atherosclerosis; coronary; genes; genetics; death; sudden
6.  Body Mass Index is Associated with USF1 Haplotype in Korean Premenopausal Women 
The upstream stimulatory factor 1 (USF1) gene has been shown to play an essential role as the cause of familial combined hyperlipidemia, and there are several association studies on the relationship between USF1 and metabolic disorders. In this study, we analyzed two single nucleotide polymorphisms in USF1 rs2073653 (306A>G) and rs2516840 (1748C>T) between the case (dyslipidemia or obesity) group and the control group in premenopausal females, postmenopausal females, and males among 275 Korean subjects. We observed a statistically significant difference in the GC haplotype between body mass index (BMI) ≥25 kg/m2 and BMI <25 kg/m2 groups in premenopausal females (χ2=4.23, p=0.04). It seems that the USF1 GC haplotype is associated with BMI in premenopausal Korean females.
doi:10.3346/jkms.2008.23.1.83
PMCID: PMC2526481  PMID: 18303204
USF1; Polymorphism; Haplotypes; Body Mass Index
7.  Pharmacogenetic association of the APOA1/C3/A4/A5 gene cluster and lipid responses to fenofibrate: the Genetics of Lipid-Lowering Drugs and Diet Network study 
Pharmacogenetics and genomics  2009;19(2):161-169.
Background
The apolipoproteins (APOA1/C3/A4/A5) are key components in modulating lipoprotein metabolism. It is unknown whether variants at the APOA1/C3/A4/A5 gene cluster are associated with lipid response to pharmacologic intervention.
Methods and results
Plasma triglycerides (TGs) and high-density lipoprotein (HDL) levels were measured in 861 Genetics of Lipid-Lowering Drugs and Diet Network study participants who underwent a 3-week fenofibrate trial. We examined 18 common single nucleotide polymorphisms (SNPs) spanning the APOA1/C3/A4/A5 genes to investigate the effects of variants at the gene cluster on lipid response to fenofibrate treatment. We found that the minor alleles of the SNPs rs3135506 (APOA5_S19W), rs5104 (APOA4_N147S), rs4520 (APOC3_G34G), and rs5128 (APOC3_3U386) were associated with enhanced TG response to fenofibrate treatment (P = 0.0004–0.018). The minor allele of SNP rs2854117 (APOC3_M482) was associated with reduced rather than enhanced TG response (P = 0.026). The SNP rs3135506 (APOA5_S19W) was associated with HDL response, with minor allele related to reduced HDL response to fenofibrate (P = 0.002). Association analyses on haplotype provided corroborative evidence to single SNP association analyses. The common haplotypes H2, H3, and H5 were significantly associated with reduced TG response to fenofibrate.
Conclusion
The genetic variants at APOA1/C3/A4/A5 gene cluster may be useful markers to predict response of lipid-lowering therapy with fenofibrate. Further studies to replicate/confirm our findings are warranted.
doi:10.1097/FPC.0b013e32831e030e
PMCID: PMC2733171  PMID: 19057464
apolipoproteins gene cluster; fenofibrate; hypertriglyceridemia; pharmacogenetics
8.  Human Apolipoprotein A-I Natural Variants: Molecular Mechanisms Underlying Amyloidogenic Propensity 
PLoS ONE  2012;7(8):e43755.
Human apolipoprotein A-I (apoA-I)-derived amyloidosis can present with either wild-type (Wt) protein deposits in atherosclerotic plaques or as a hereditary form in which apoA-I variants deposit causing multiple organ failure. More than 15 single amino acid replacement amyloidogenic apoA-I variants have been described, but the molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here, we have investigated by fluorescence and biochemical approaches the stabilities and propensities to aggregate of two disease-associated apoA-I variants, apoA-IGly26Arg, associated with polyneuropathy and kidney dysfunction, and apoA-ILys107-0, implicated in amyloidosis in severe atherosclerosis. Results showed that both variants share common structural properties including decreased stability compared to Wt apoA-I and a more flexible structure that gives rise to formation of partially folded states. Interestingly, however, distinct features appear to determine their pathogenic mechanisms. ApoA-ILys107-0 has an increased propensity to aggregate at physiological pH and in a pro-inflammatory microenvironment than Wt apoA-I, whereas apoA-IGly26Arg elicited macrophage activation, thus stimulating local chronic inflammation. Our results strongly suggest that some natural mutations in apoA-I variants elicit protein tendency to aggregate, but in addition the specific interaction of different variants with macrophages may contribute to cellular stress and toxicity in hereditary amyloidosis.
doi:10.1371/journal.pone.0043755
PMCID: PMC3429494  PMID: 22952757
9.  Apolipoprotein A-V dependent modulation of plasma triacylglycerol: A puzzlement☆,☆☆ 
Biochimica et Biophysica Acta  2011;1821(5):795-799.
The discovery of apolipoprotein A-V (apoA-V) in 2001 has raised a number of intriguing questions about role in lipid transport and triglyceride (TG) homeostasis. Genome wide association studies (GWAS) have consistently identified APOA5 as a contributor to plasma TG levels. Single nucleotide polymorphisms (SNP) with-in the APOA5 gene locus have been shown to correlate with elevated plasma TG. Furthermore, transgenic and knockout mouse models support the view that apoA-V plays a critical role in maintenance of plasma TG levels. The present review describes recent concepts pertaining to apoA-V SNP analysis and their association with elevated plasma TG. The interaction of apoA-V with glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) is discussed relative to its postulated role in TG-rich lipoprotein catabolism. The potential role of intracellular apoA-V in regulation of TG homeostasis, as a function of its ability to associate with cytosolic lipid droplets, is reviewed. While some answers are emerging, numerous mysteries remain with regard to this low abundance, yet potent, modulator of TG homeostasis. Given the strong correlation between elevated plasma TG and heart disease, there is great scientific and public interest in deciphering the numerous biological riddles presented by apoA-V. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.
doi:10.1016/j.bbalip.2011.12.002
PMCID: PMC3319174  PMID: 22209939
Apolipoprotein A-V; Triglyceride; Lipid droplet; Genome wide association study; Glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1; Nonalcoholic fatty liver disease
10.  Upstream Transcription Factor 1 (USF1) allelic variants regulate lipoprotein metabolism in women and USF1 expression in atherosclerotic plaque 
Scientific Reports  2014;4:4650.
Upstream transcription factor 1 (USF1) allelic variants significantly influence future risk of cardiovascular disease and overall mortality in females. We investigated sex-specific effects of USF1 gene allelic variants on serum indices of lipoprotein metabolism, early markers of asymptomatic atherosclerosis and their changes during six years of follow-up. In addition, we investigated the cis-regulatory role of these USF1 variants in artery wall tissues in Caucasians. In the Cardiovascular Risk in Young Finns Study, 1,608 participants (56% women, aged 31.9 ± 4.9) with lipids and cIMT data were included. For functional study, whole genome mRNA expression profiling was performed in 91 histologically classified atherosclerotic samples. In females, serum total, LDL cholesterol and apoB levels increased gradually according to USF1 rs2516839 genotypes TT < CT < CC and rs1556259 AA < AG < GG as well as according to USF1 H3 (GCCCGG) copy number 0 < 1 < 2. Furthermore, the carriers of minor alleles of rs2516839 (C) and rs1556259 (G) of USF1 gene had decreased USF1 expression in atherosclerotic plaques (P = 0.028 and 0.08, respectively) as compared to non-carriers. The genetic variation in USF1 influence USF1 transcript expression in advanced atherosclerosis and regulates levels and metabolism of circulating apoB and apoB-containing lipoprotein particles in sex-dependent manner, but is not a major determinant of early markers of atherosclerosis.
doi:10.1038/srep04650
PMCID: PMC3983598  PMID: 24722012
11.  Associations among Race/Ethnicity, ApoC-III Genotypes, and Lipids in HIV-1-Infected Individuals on Antiretroviral Therapy 
PLoS Medicine  2006;3(3):e52.
Background
Protease inhibitors (PIs) are associated with hypertriglyceridemia and atherogenic dyslipidemia. Identifying HIV-1-infected individuals who are at increased risk of PI-related dyslipidemia will facilitate therapeutic choices that maintain viral suppression while reducing risk of atherosclerotic diseases. Apolipoprotein C-III (apoC-III) gene variants, which vary by race/ethnicity, have been associated with a lipid profile that resembles PI-induced dyslipidemia. However, the association of race/ethnicity, or candidate gene effects across race/ethnicity, with plasma lipid levels in HIV-1-infected individuals, has not been reported.
Methods and Findings
A cross-sectional analysis of race/ethnicity, apoC-III/apoA-I genotypes, and PI exposure on plasma lipids was performed in AIDS Clinical Trial Group studies (n = 626). Race/ethnicity was a highly significant predictor of plasma lipids in fully adjusted models. Furthermore, in stratified analyses, the effect of PI exposure appeared to differ across race/ethnicity. Black/non-Hispanic, compared with White/non-Hispanics and Hispanics, had lower plasma triglyceride (TG) levels overall, but the greatest increase in TG levels when exposed to PIs. In Hispanics, current PI antiretroviral therapy (ART) exposure was associated with a significantly smaller increase in TGs among patients with variant alleles at apoC-III-482, −455, and Intron 1, or at a composite apoC-III genotype, compared with patients with the wild-type genotypes.
Conclusions
In the first pharmacogenetic study of its kind in HIV-1 disease, we found race/ethnic-specific differences in plasma lipid levels on ART, as well as differences in the influence of the apoC-III gene on the development of PI-related hypertriglyceridemia. Given the multi-ethnic distribution of HIV-1 infection, our findings underscore the need for future studies of metabolic and cardiovascular complications of ART that specifically account for racial/ethnic heterogeneity, particularly when assessing candidate gene effects.
This cross-sectional analysis of race/ethnicity, apoC-III/apoA-I genotypes, and protease inhibitor exposure on plasma lipids showed that race/ethnicity was a highly significant predictor of plasma lipids
doi:10.1371/journal.pmed.0030052
PMCID: PMC1334223  PMID: 16417409
12.  Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor▿  
Molecular and Cellular Biology  2009;29(21):5900-5910.
Transcription factor USF is a ubiquitously expressed member of the helix-loop-helix family of proteins. It binds with high affinity to E-box elements and, through interaction with coactivators, aids in the formation of transcription complexes. Previous work demonstrated that USF regulates genes during erythroid differentiation, including HoxB4 and β-globin. Here, we show that the erythroid cell-specific expression of a dominant-negative mutant of USF, A-USF, in transgenic mice reduces the expression of all β-type globin genes and leads to the diminished association of RNA polymerase II with locus control region element HS2 and with the β-globin gene promoter. We further show that the expression of A-USF reduces the expression of several key erythroid cell-specific transcription factors, including EKLF and Tal-1. We provide evidence demonstrating that USF interacts with known regulatory DNA elements in the EKLF and Tal-1 gene loci in erythroid cells. Furthermore, A-USF-expressing transgenic mice exhibit a defect in the formation of CD71+ progenitor and Ter-119+ mature erythroid cells. In summary, the data demonstrate that USF regulates globin gene expression indirectly by enhancing the expression of erythroid transcription factors and directly by mediating the recruitment of transcription complexes to the globin gene locus.
doi:10.1128/MCB.00419-09
PMCID: PMC2772736  PMID: 19704006
13.  Associations of Polymorphisms in the Apolipoprotein APOA1-C3-A5 Gene Cluster with Acute Coronary Syndrome 
Background. Acute coronary syndromes (ACSs) are clinically cardiovascular events associated with dyslipidemia in common. Single nucleotide polymorphisms (SNPs) and haplotypes in the APOA1/C3/A5 gene cluster are associated with diabetes and familial combined hyperlipidaemia (FCH). Little is known about whether the polymorphisms in these genes affect lipid homeostasis in patients with ACSs. The present paper aimed to examine these associations with 4 SNPs in the APOA1 −75G > A, the APOC3 −455T > C, and APOA5 −1131T > C, c.553G > T variant to ACSs in Chinese Han. Methods. Chinese Han of 229 patients with ACSs and 254 unrelated controls were analyzed. Four SNPs in APOA1/C3/A5 cluster were genotyped and lipid was determined. Results. Our data show that minor allelic frequencies of APOC3 −455T > C, APOA5 −1131T > C, and c.553G > T polymorphisms in patients with ACSs were significantly higher than control group (P < 0.05). Furthermore, the 3 polymorphic sites were strongly of linkage disequilibrium, and minor alleles of 3 SNP sites had higher TG level than wild alleles (P < 0.05), APOC3 −455C and APOA5 c.553T allele carriers also had lower level of HDL-C. Conclusions. The minor alleles of APOC3 −455T > C, APOA5 −1131T > C, and c.553G > T polymorphisms are closely associated with ACSs.
doi:10.1155/2012/509420
PMCID: PMC3366243  PMID: 22675253
14.  Determinants of Plasma Apolipoprotein A-V and APOA5 Gene Transcripts in Humans 
Journal of internal medicine  2008;264(5):452-462.
Objective
Apolipoprotein A-V (apoAV) contributes to the regulation of triglyceride metabolism, which plays a role in the pathogenesis of atherosclerotic diseases. We therefore ascertained determinants of hepatic APOA5 transcript and apoAV plasma levels in humans.
Design
We determined influences of anthropometric variables, biochemical factors related to lipid and glucose metabolism, hepatic mRNA levels transcribed from the APOA1/C3/A4/A5 cluster and transcription factor genes implicated in the regulation of APOA5 as well as common SNPs at the APOA5 locus on APOA5 expression in 89 obese patients and 22 non-obese controls.
Results
Mean, age and sex adjusted, hepatic APOA5 mRNA or apoAV plasma levels did not differ by obesity status, HOMA-IR or inflammatory markers. In multivariate regression models, the c56C>G SNP, plasma apoCIII, plasma non-esterified fatty acids, hepatic APOA5 transcripts, sex and a weak association with obesity status explained 61% of the variance in apoAV plasma levels. Hepatic transcript levels of CPT1A1 and PPARA, plasma non-esterified fatty acids and the c56C>G SNP explained 48% of the variance in hepatic APOA5 transcript levels.
Conclusion
ApoAV plasma levels are independently associated with plasma free fatty acid and hepatic APOA5 mRNA levels. Associations of APOA5 transcripts with PPARA and CPT1A1 transcripts suggest that APOA5 expression is intimately linked to hepatic lipid metabolism.
doi:10.1111/j.1365-2796.2008.01987.x
PMCID: PMC3533125  PMID: 18537870
Apolipoprotein A-V; APOA5; triglycerides; PPARA; CPT1A; SNP
15.  Overexpression of USF Increases TGF-β1 Protein Levels, But G1 Phase Arrest was not Induced in FRTL-5 Cells 
Journal of Korean Medical Science  2008;23(5):870-876.
Transforming growth factor-β1 (TGF-β1) is a potent inhibitor of cellular growth and proliferation by G1 phase arrest or apoptosis. We investigated the association of TGF-β1 with the anti-proliferative effect of upstream stimulatory factor (USF) in Fischer rat thyroid cell line (FRTL-5) cells. [Methyl-3H] thymidine uptake was measured after treatment of FRTL-5 cells with TGF-β1 to identify its anti-proliferative effect. USF-1 and USF-2 proteins were in vitro translated, and an electrophoretic mobility shift assay was performed to identify the interaction between USF and the TGF-β1 promoter. FRTL-5 cells were transfected with USF cDNA, and then the expression of TGF-β1 was examined with Northern and Western blotting. The cell cycle-regulating proteins associated with TGF-β1 were also measured. TGF-β1 significantly inhibited [methyl-3H] thymidine uptake in FRTL-5 cells. Two specific binding sites for USF were found in the TGF-β1 promoter: -1,846~-1,841 (CACATG) and -621~-616 (CATGTG). Overexpression of USF increased both the mRNA levels and protein levels of TGF-β1. However, the expression of cyclin D1, CDK4, cyclin E, and CDK2, and the phosphorylation of retinoblastoma protein remained unchanged. Overexpression of USF in FRTL-5 cells increased the expression of TGF-β10 through specific binding to TGF-β1 promoter. However, the USF-induced expression of TGF-β1 did not cause G1 arrest.
doi:10.3346/jkms.2008.23.5.870
PMCID: PMC2580023  PMID: 18955796
Upstream Stimulatory Factor; Transforming Growth Factor beta1; FRTL-5 Cells
16.  A Systems Genetics Approach Implicates USF1, FADS3, and Other Causal Candidate Genes for Familial Combined Hyperlipidemia 
PLoS Genetics  2009;5(9):e1000642.
We hypothesized that a common SNP in the 3' untranslated region of the upstream transcription factor 1 (USF1), rs3737787, may affect lipid traits by influencing gene expression levels, and we investigated this possibility utilizing the Mexican population, which has a high predisposition to dyslipidemia. We first associated rs3737787 genotypes in Mexican Familial Combined Hyperlipidemia (FCHL) case/control fat biopsies, with global expression patterns. To identify sets of co-expressed genes co-regulated by similar factors such as transcription factors, genetic variants, or environmental effects, we utilized weighted gene co-expression network analysis (WGCNA). Through WGCNA in the Mexican FCHL fat biopsies we identified two significant Triglyceride (TG)-associated co-expression modules. One of these modules was also associated with FCHL, the other FCHL component traits, and rs3737787 genotypes. This USF1-regulated FCHL-associated (URFA) module was enriched for genes involved in lipid metabolic processes. Using systems genetics procedures we identified 18 causal candidate genes in the URFA module. The FCHL causal candidate gene fatty acid desaturase 3 (FADS3) was associated with TGs in a recent Caucasian genome-wide significant association study and we replicated this association in Mexican FCHL families. Based on a USF1-regulated FCHL-associated co-expression module and SNP rs3737787, we identify a set of causal candidate genes for FCHL-related traits. We then provide evidence from two independent datasets supporting FADS3 as a causal gene for FCHL and elevated TGs in Mexicans.
Author Summary
By integrating a genetic polymorphism with genome-wide gene expression levels, we were able to attribute function to a genetic polymorphism in the USF1 gene. The USF1 gene has previously been associated with a common dyslipidemia, FCHL. FCHL is characterized by elevated levels of total cholesterol, triglycerides, or both. We demonstrate that this genetic polymorphism in USF1 contributes to FCHL disease risk by modulating the expression of a group of genes functionally related to lipid metabolism, and that this modulation is mediated by USF1. One of the genes whose expression is modulated by USF1 is FADS3, which was also implicated in a recent genome-wide association study for lipid traits. We demonstrated that a genetic polymorphism from the FADS3 region, which was associated with triglycerides in a GWAS study of Caucasians, was also associated with triglycerides in Mexican FCHL families. Our analysis provides novel insight into the gene expression profile contributing to FCHL disease risk, and identifies FADS3 as a new gene for FCHL in Mexicans.
doi:10.1371/journal.pgen.1000642
PMCID: PMC2730565  PMID: 19750004
17.  Apolipoprotein A5 and lipoprotein lipase interact to modulate anthropometric measures in Hispanics of Caribbean origin 
Apolipoprotein A5 (APOA5) and lipoprotein lipase (LPL) proteins interact functionally to regulate lipid metabolism, and single nucleotide polymorphisms (SNPs) for each gene have also been associated independently with obesity risk. Evaluating gene combinations may be more effective than single SNP analyses in identifying genetic risk, but insufficient minor allele frequency (MAF) often limits evaluations of potential epistatic relationships. Populations with multiple ancestral admixtures may provide unique opportunities for evaluating genetic interactions. We examined relationships between LPL m107 (rs1800590) and APOA5 S19W (rs3135506) and lipid and anthropometric measures in Caribbean origin Hispanics (n=1019, aged 45–5 years) living in the Boston metropolitan area. Significant interaction terms between LPL m107 and APOA5 S19W were observed for BMI (P=0.003) and waist circumference (P=0.019). Higher BMI (P=0.001), waist (P=0.011) and hip (P=0.026) were observed in minor allele (G) carriers for LPL m107 who also carried the APOA5 S19W minor allele (G). Additionally, extreme obesity (BMI≥40 kg/m2) risk was higher (OR=4.02; 95% CI:1.81–.91; global P=0.008) for minor allele carriers for both SNPs (LPL TG+GG, APOA5 CG+GG) compared to major allele carriers for both SNPs. In summary, we identified significant interactions for APOA5 S19W and LPL m107 for obesity in Caribbean Hispanics. Population-specific MAFs increase the difficulties of replicating gene-gene interactions, but may support the hypothesis that combinations of frequencies in selected genes could heighten obesity susceptibility in a given population. Analyses of gene-gene interactions may improve understanding of genetically-based obesity risk, and underscore the need for further study of groups with multiple ancestral admixtures.
doi:10.1038/oby.2009.216
PMCID: PMC2813926  PMID: 19629056
18.  Participation of upstream stimulator factor (USF) in cadmium-induction of the mouse metallothionein-I gene. 
Nucleic Acids Research  1998;26(22):5182-5189.
The roles of the bHLH-Zip protein, upstream stimulatory factor (USF), in mouse metallothionein-I (MT-I) gene expression were examined. The promoter contains a putative USF binding site which overlaps an antioxidant response element (ARE) located at -101 bp relative to the transcription start point. The USF/ARE composite element increases basal expression of the mouse MT-I gene, and partly mediates response to oxidative stress. However, other functions of this composite element and the in vivo roles for USF in MT-I promoter functions have not been examined. We report studies which indicate that USF participates via the USF/ARE element in cadmium responsiveness of the mouse MT-I promoter. During the course of these studies a second, higher affinity USF binding site at -223 bp was identified. Stable and transient transfection assays in mouse hepatoma cells, using the USF/ARE in the context of a minimal promoter and site-directed and truncation mutants of the MT-I promoter, revealed that the USF and the ARE sites contribute to cadmium (2-30 microM) but not zinc responsiveness, and to basal promoter activity. Overexpression of dominant-negative (dn)USF in co-transfection assays significantly attenuated cadmium induction of the USF/ARE in the context of a minimal promoter, and attenuated cadmium, but not zinc, induction of the intact MT-I promoter. A consensus E-box (CACATG) at -223 bp in the MT-I promoter was also found to bind USF in vitro , and to be constitutively footprinted in vivo . The interaction of USF with E-box1 was apparently 10-fold stronger than that with the USF/ARE. However, in contrast, E-box1 was not a strong basal promoter element nor was it metal ions responsive in mouse Hepa cells. In conclusion, these studies demonstrate a role for USF in cadmium-specific induction of the mouse MT-I gene, but bring into question an obligate role for USF in regulating basal activity of this gene. The data further suggest that USF interacts with ARE-binding proteins to influence MT-I gene expression.
PMCID: PMC147950  PMID: 9801317
19.  Apolipoprotein A-V associates with intrahepatic lipid droplets and influences triglyceride accumulation 
Biochimica et biophysica acta  2010;1801(5):605-608.
Apolipoprotein A-V (apoA-V), secreted solely by the liver, is a low abundance protein that strongly influences plasma triglyceride (TG) levels. In vitro, in transfected hepatoma cell lines apoA-V is largely retained within the cell in association with cytosolic lipid droplets (LD). To evaluate if this is true in vivo, in the present study the amount of apoA-V in the plasma compartment versus liver tissue was determined in APOA5 transgenic (Tg) mice. The majority of total apoA-V (~80%) was in the plasma compartment. Injection of APOA5 Tg mice with heparin increased plasma apoA-V protein levels by ~25% indicating the existence of a heparin-releasable pool. Intrahepatic apoA-V was associated with LD isolated from livers of wild type (WT) and APOA5 Tg mice. Furthermore, livers from APOA5 Tg mice contained significantly higher amounts of TG than livers from WT or apoa5 knockout mice suggesting the apoA-V influences intrahepatic TG levels.
doi:10.1016/j.bbalip.2010.02.004
PMCID: PMC2839894  PMID: 20153840
apolipoprotein A-V; APOA5 transgenic mice; liver; lipid droplets; triglyceride
20.  Linkage and Association Between Distinct Variants of the APOA1/C3/A4/A5 Gene Cluster and Familial Combined Hyperlipidemia 
Objective
Combined hyperlipidemia is a common disorder, characterized by a highly atherogenic lipoprotein profile and a substantially increased risk of coronary heart disease. The purpose of this study was to establish whether variations of apolipoprotein A5 (APOA5), a newly discovered gene of lipid metabolism located 30 kbp downstream of the APOA1/C3/A4 gene cluster, contributes to the transmission of familial combined hyperlipidemia (FCHL).
Methods and Results
We performed linkage and association tests on 128 families. Two independent alleles, APOA5c.56G and APOC3c.386G, of the APOA1/C3/A4/A5 gene cluster were overtransmitted in FCHL (P=0.004 and 0.007, respectively). This was paired with reduced transmission of the common APOA1/C3/A4/A5 haplotype (frequency 0.4461) to affected subjects (P=0.012). The APOA5c.56G genotype accounted for 7.3% to 13.8% of the variance in plasma triglyceride levels in probands (P<0.004). The APOC3c.386G genotypes accounted for 4.4% to 5.1% of the variance in triglyceride levels in FCHL spouses (P<0.007), suggesting that this allele marks a FCHL quantitative trait as well as representing a susceptibility locus for the condition.
Conclusions
A combined linkage and association analysis establishes that variation at the APOA1/C3/A4/A5 gene cluster contributes to FCHL transmission in a substantial proportion of northern European families.
doi:10.1161/01.ATV.0000099881.83261.D4
PMCID: PMC2773540  PMID: 14551155
apolipoproteins; genes; risk factors; genetics; hyperlipoproteinemia
21.  Triglyceride associated polymorphisms of the APOA5 gene have very different allele frequencies in Pune, India compared to Europeans 
BMC Medical Genetics  2006;7:76.
Background
The APOA5 gene variants, -1131T>C and S19W, are associated with altered triglyceride concentrations in studies of subjects of Caucasian and East Asian descent. There are few studies of these variants in South Asians. We investigated whether the two APOA5 variants also show similar association with various lipid parameters in Indian population as in the UK white subjects.
Methods
We genotyped 557 Indian adults from Pune, India, and 237 UK white adults for -1131T>C and S19W variants in the APOA5 gene, compared their allelic and genotype frequency and determined their association with fasting serum triglycerides, total cholesterol, HDL and LDL cholesterol levels using univariate general linear analysis. APOC3 SstI polymorphism was also analyzed in 175 Pune Indian subjects for analysis of linkage disequilibrium with the APOA5 variants.
Results
The APOA5 -1131C allele was more prevalent in Indians from Pune (Pune Indians) compared to UK white subjects (allele frequency 20% vs. 4%, p = 0.00001), whereas the 19W allele was less prevalent (3% vs. 6% p = 0.0015). Patterns of linkage disequilibrium between the two variants were similar between the two populations and confirmed that they occur on two different haplotypes. In Pune Indians, the presence of -1131C allele and the 19W allele was associated with a 19% and 15% increase respectively in triglyceride concentrations although only -1131C was significant (p = 0.0003). This effect size was similar to that seen in the UK white subjects. Analysis of the APOC3 SstI polymorphism in 175 Pune Indian subjects showed that this variant is not in appreciable linkage disequilibrium with the APOA5 -1131T>C variant (r2 = 0.07).
Conclusion
This is the first study to look at the role of APOA5 in Asian Indian subjects that reside in India. The -1131C allele is more prevalent and the 19W allele is less prevalent in Pune Indians compared to UK Caucasians. We confirm that the APOA5 variants are associated with triglyceride levels independent of ethnicity and that this association is similar in magnitude in Asian Indians and Caucasians. The -1131C allele is present in 36% of the Pune Indian population making it a powerful marker for looking at the role of elevated triglycerides in important conditions such as pancreatitis, diabetes and coronary heart disease.
doi:10.1186/1471-2350-7-76
PMCID: PMC1618828  PMID: 17032446
22.  The Interaction of ApoA-I and ABCA1 Triggers Signal Transduction Pathways to Mediate Efflux of Cellular Lipids 
Molecular Medicine  2011;18(1):149-158.
Reverse cholesterol transport (RCT) has been characterized as a crucial step for antiatherosclerosis, which is initiated by ATP-binding cassette A1 (ABCA1) to mediate the efflux of cellular phospholipids and cholesterol to lipid-free apolipoprotein A-I (apoA-I). However, the mechanisms underlying apoA-I/ABCA1 interaction to lead to the lipidation of apoA-I are poorly understood. There are several models proposed for the interaction of apoA-I with ABCA1 as well as the lipidation of apoA-I mediated by ABCA1. ApoA-I increases the levels of ABCA1 protein markedly. In turn, ABCA1 can stabilize apoA-I. The interaction of apoA-I with ABCA1 could activate signaling molecules that modulate posttranslational ABCA1 activity or lipid transport activity. The key signaling molecules in these processes include protein kinase A (PKA), protein kinase C (PKC), Janus kinase 2 (JAK2), Rho GTPases and Ca2+, and many factors also could influence the interaction of apoA-I with ABCA1. This review will summarize these mechanisms for the apoA-I interaction with ABCA1 as well as the signal transduction pathways involved in these processes.
doi:10.2119/molmed.2011.00183
PMCID: PMC3320140  PMID: 22064972
23.  Influence of ApoA-I Domain Structure on Macrophage Reverse Cholesterol Transport in Mice 
Objective
To determine the influence of apoA-I tertiary structure domain properties on the anti-atherogenic properties of the protein. Two chimeric hybrids with the N-terminal domains swapped (human-M apoA-I and mouse-H apoA-I) were expressed in apoA-I-null mice with adeno-associated virus (AAV) and used to study macrophage reverse cholesterol transport (RCT) in vivo.
Methods and Results
The different apoA-I variants were expressed in apoA-I-null mice that were injected with [H3]cholesterol-labeled J774 mouse macrophages to measure RCT. Significantly more cholesterol was removed from the macrophages and deposited in the feces via the RCT pathway in mice expressing mouse-H apoA-I compared to all other groups. Analysis of the individual components of the RCT pathway demonstrated that mouse-H apoA-I promoted ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux more efficiently than all other variants as well as increasing the rate of cholesterol uptake into liver cells.
Conclusion
The structural domain properties of apoA-I affect the ability of the protein to mediate macrophage RCT. Substitution of the N-terminal helix bundle domain in the human apoA-I with the mouse apoA-I counterpart causes a gain of function with respect to macrophage RCT, suggesting that engineering some destabilization into the N-terminal helix bundle domain and/or increasing the hydrophobicity of the C-terminal domain of human apoA-I would enhance the anti-atherogenic properties of the protein.
doi:10.1161/ATVBAHA.110.216226
PMCID: PMC3024460  PMID: 21071688
apoA-I; reverse cholesterol transport; high density lipoprotein; macrophage; apolipoprotein
24.  Consumption of whole grains and legumes modulates the genetic effect of the APOA5 -1131C variant on changes in triglyceride and apolipoprotein A-V concentrations in patients with impaired fasting glucose or newly diagnosed type 2 diabetes 
Trials  2014;15:100.
Background
The apolipoprotein A5 gene (APOA5) -1131 T > C polymorphism is associated with mild hypertriglyceridemia in type 2 diabetic subjects, and interacts with dietary fat in the determination of triglyceride concentrations. We examined whether a substitution of whole grains and legumes for refined rice in a high carbohydrate diet (about 65% of energy derived from carbohydrate) may modify the effect of this variant on changes in apolipoprotein A-V (apoA-V) and triglyceride concentrations.
Methods
We genotyped the APOA5 -1131 T > C in individuals with impaired fasting glucose (IFG) or newly diagnosed type 2 diabetes, who were randomly assigned to either a group ingesting whole grain and legume meals daily or a control group for 12 weeks.
Results
After dietary intervention, we observed significant interactions between the APOA5 -1131 T > C polymorphism and carbohydrate sources (whole grains and legumes versus refined rice) in the determination of mean percent changes in triglyceride and apoA-V (P interactions <0.001 and =0.038, respectively). In the refined rice group (n = 93), the carriers of the risk C allele (n = 50) showed a greater increase in the mean percent changes of triglyceride and apoA-V than noncarriers after adjusting for HOMA-IR (P = 0.004 and 0.021, respectively). The whole grain and legume group (n = 92), however, showed a decrease in fasting glucose, HOMA-IR, and triglyceride, and an increase in apoA-V, irrespective of genotype.
Conclusions
The data showed that the magnitude of the genetic effect of the APOA5 -1131C variant on triglyceride and apoA-V levels was modulated when substituting consumption of whole grains and legumes for refined rice as a carbohydrate source in IFG or diabetic subjects.
Trial registration
ClinicalTrials.gov: NCT01784952.
doi:10.1186/1745-6215-15-100
PMCID: PMC3974230  PMID: 24690159
APOA5 -1131 T > C; Whole grains and legumes; Triglycerides; Apolipoprotein A-V
25.  Additive effects of LPL, APOA5 and APOE variant combinations on triglyceride levels and hypertriglyceridemia: results of the ICARIA genetic sub-study 
BMC Medical Genetics  2010;11:66.
Background
Hypertriglyceridemia (HTG) is a well-established independent risk factor for cardiovascular disease and the influence of several genetic variants in genes related with triglyceride (TG) metabolism has been described, including LPL, APOA5 and APOE. The combined analysis of these polymorphisms could produce clinically meaningful complementary information.
Methods
A subgroup of the ICARIA study comprising 1825 Spanish subjects (80% men, mean age 36 years) was genotyped for the LPL-HindIII (rs320), S447X (rs328), D9N (rs1801177) and N291S (rs268) polymorphisms, the APOA5-S19W (rs3135506) and -1131T/C (rs662799) variants, and the APOE polymorphism (rs429358; rs7412) using PCR and restriction analysis and TaqMan assays. We used regression analyses to examine their combined effects on TG levels (with the log-transformed variable) and the association of variant combinations with TG levels and hypertriglyceridemia (TG ≥ 1.69 mmol/L), including the covariates: gender, age, waist circumference, blood glucose, blood pressure, smoking and alcohol consumption.
Results
We found a significant lowering effect of the LPL-HindIII and S447X polymorphisms (p < 0.0001). In addition, the D9N, N291S, S19W and -1131T/C variants and the APOE-ε4 allele were significantly associated with an independent additive TG-raising effect (p < 0.05, p < 0.01, p < 0.001, p < 0.0001 and p < 0.001, respectively). Grouping individuals according to the presence of TG-lowering or TG-raising polymorphisms showed significant differences in TG levels (p < 0.0001), with the lowest levels exhibited by carriers of two lowering variants (10.2% reduction in TG geometric mean with respect to individuals who were homozygous for the frequent alleles of all the variants), and the highest levels in carriers of raising combinations (25.1% mean TG increase). Thus, carrying two lowering variants was protective against HTG (OR = 0.62; 95% CI, 0.39-0.98; p = 0.042) and having one single raising polymorphism (OR = 1.20; 95% CI, 1.39-2.87; p < 0.001) or more (2 or 3 raising variants; OR = 2.90; 95% CI, 1.56-5.41; p < 0.001) were associated with HTG.
Conclusion
Our results showed a significant independent additive effect on TG levels of the LPL polymorphisms HindIII, S447X, D9N and N291S; the S19W and -1131T/C variants of APOA5, and the ε4 allele of APOE in our study population. Moreover, some of the variant combinations studied were significantly associated with the absence or the presence of hypertriglyceridemia.
doi:10.1186/1471-2350-11-66
PMCID: PMC2877669  PMID: 20429872

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