Sotirios K. Karathanasis

The Role of CBP in Estrogen Receptor Cross-Talk with Nuclear Factor-κB in HepG2 Cells

Functional interactions or cross-talk between ligand-activated nuclear receptors and the proinflammatory transcription factor nuclear factor-κB (NF-κB) may play a major role in ligand-mediated modification of diseases processes. In particular, the cardioprotective effects of estrogen replacement therapy are thought to be due in part to the ability of ligand-bound estrogen receptor (ER) to inhibit NF-κB function. In the current study 17β-estradiol-bound ERα interfered with cytokine-induced activation of a NF-κB reporter in HepG2 cells. The estrogen metabolite, 17α-ethinyl estradiol, and the phytoestrogen, genistein, were also effective inhibitors of NF-κB activation, whereas tamoxifen, 4-hydroxytamoxifen, and raloxifene were inactive. This inhibition was reciprocal, as NF-κB interfered with the trans-activation properties of ERα. Ligand-bound ERα did not inhibit NF-κB binding to DNA, but it did decrease the histone acetyltransferase activity required for NF-κB transcriptional activity. Coexpression of the ...

Estrogen Regulation of the Apolipoprotein AI Gene Promoter through Transcription Cofactor Sharing

Estrogen replacement therapy increases plasma concentrations of high density lipoprotein and its major protein constituent, apolipoprotein AI (apoAI). Studies with animal model systems, however, suggest opposite effects. In HepG2 cells stably expressing estrogen receptor α (ERα), 17β-estradiol (E2) potently inhibited apoAI mRNA steady state levels. ApoAI promoter deletion mapping experiments indicated that ERα plus E2 inhibited apoAI activity through the liver-specific enhancer. Although the ERα DNA binding domain was essential but not sufficient for apoAI enhancer inhibition, ERα binding to the apoAI enhancer could not be detected by electrophoretic mobility shift assays. Western blotting and cotransfection assays showed that ERα plus E2 did not influence the abundance or the activity of the hepatocyte-enriched factors HNF-3β and HNF-4, two transcription factors essential for apoAI enhancer function. Expression of the ERα coactivator RIP140 dramatically repressed apoAI enhancer function in cotransfection experiments, suggesting that RIP140 may also function as a coactivator on the apoAI enhancer. Moreover, estrogen regulation of apoAI enhancer activity was dependent upon the balance between ERα and RIP140 levels. At low ratios of RIP140 to ERα, E2 repressed apoAI enhancer activity, whereas at high ratios this repression was reversed. Regulation of the apoAI gene by estrogen may thus vary in direction and magnitude depending not only on the presence of ERα and E2 but also upon the intracellular balance of ERα and coactivators utilized by ERα and the apoAI enhancer.

Genetic linkage of the human apolipoprotein AI-CIII-AIV gene cluster and the neural cell adhesion molecule (NCAM) gene

The genes encoding apolipoproteins AI, CIII, and AIV, three plasma proteins involved in lipid metabolism, are clustered within a 15-kb DNA segment (apoAI-CIII-AIV gene cluster) located on human chromosome 11 at band q23. The gene encoding the neural cell adhesion molecule (NCAM), a cell surface glycoprotein involved in cell-cell recognition during morphogenesis, is also located on chromosome 11, band q23. In this report, 12 previously described restriction fragment length polymorphisms (RFLPs) in the apoAI-CIII-AIV gene cluster were tested for cosegregation with a newly identified BamHI RFLP in the NCAM gene using 13 families. The results show that the apoAI-CIII-AIV gene cluster and the NCAM gene loci are linked with a maximum lod score of 15.9 at a recombination fraction of 0.028. In addition, an approach for the most efficient use of the apoAI-CIII-AIV gene cluster polymorphisms, based on the evaluation of their individual and cumulative heterozygosities, is presented.

Apolipoprotein A1 Baltimore (Arg10→Leu), a new ApoA1 variant

SummaryA new apolipoprotein A1 (APOA1) gene variant has been identified in a family ascertained through a proband undergoing coronary angiography. The variant, ApoA1 Baltimore, was due to a mutation at codon 34 of the third exon of the APOA1 gene (CGA to CTA) that resulted in an arginine-to-leucine substitution at the tenth amino acid of the mature ApoA1 and a change in charge of -1. The mutation abolishes a TaqI restriction site and it is easily detectable after polymerase chain reaction amplification of genomic DNA. The proband was heterozygous for the mutation. Eight other members of the pedigree had the same ApoA1 variant. Cosegregation of the variant with hypoalphalipoproteinemia could not be demonstrated and the association of this mutation with hypoalphalipoproteinemia was confined to three affected members of the nuclear family. No effect of the mutant on any lipoprotein phenotype could be established.

Expression of the Human β3-Adrenergic Receptor Gene in SK-N-MC Cells Is Under the Control of a Distal Enhancer

Mechanisms of transcriptional regulation of the humanβ 3-adrenergic receptor were studied using SK-N-MC cells, a human neuroblastoma cell line that expresses β3- andβ 1-adrenergic receptors endogenously. Deletions spanning different portions of a 7-kb 5′-flanking region of the humanβ 3-adrenergic receptor gene were linked to a luciferase reporter and transfected in SK-N-MC, CV-1, and HeLa cells. Maximal luciferase activity was observed when a 200-bp region located between− 6.5 and −6.3 kb from the translation start site was present. This region functioned only in SK-N-MC cells. Electrophoretic mobility shift assays of nuclear extracts from SK-N-MC, CV-1, and HeLa cells using double stranded oligonucleotides spanning different portions of the 200-bp region as probes and transient transfection studies revealed the existence of three cis-acting regulatory elements: A)− 6.468 kb-AGGTGGACT-−6.458 kb, B) −6.448 kb-GCCTCTCTGGGGAGCAGCTTCTCC-6.428 kb, and C) −6.405 kb-20 repeats of CCTT-6.385 kb. These elements ac...

Apolipoprotein Genes: Organization, Linkage and Evolution

In the past three years all of the genes coding for the major human apolipoproteins have been cloned and sequenced. Using these cloned DNA sequences as probes for i) genomic blotting analysis of DNA from interspecies somatic cell hybrids, i i) in situ hybridization of metaphase chromosomes, i i i) chromosomal “walking” by isolation and characterization of overlapping genomic clones, iv) genetic cosegregation of polymorphic DNA markers in family studies, and v) genomic blotting analysis of chromosomal DNA resolved by pulsed-field gel electrophoresis, it has been possible to determine the chromosomal localization and linkage relationships between all of these genes. Several conclusions have emerged. First, certain apolipoprotein genes are physically linked (i.e. clustered). For example, the apolipoprotein AI (apoAI), CIII (apoCIII) and AIV (apoAIV) genes are clustered within a 15 kilobase (kb) DNA fragment on the long arm of human chromosome 11 (1). Similarly, the apolipoprotein E (apoE), CI (apoCI) and CII (apoCII) genes are clustered within an approximately 50 kb DNA segment on the long arm of chromosome 19 (2). In contrast, the apolipoprotein AII (apoAII), B (apoB) and D (apoD) are dispersed on separate chromosomes, chromosomes 1, 2 and 3 respectively (reviewed in ref. 3).

Linkage of Human Apolipoprotein Ai, Ciii and Aiv Genes

Abstract The genes for two of the proteins of the plasma lipid transport system, apolipoprotein AI (apoAI) and CIII (apoCIII) have been shown to be closely linked in the human genome. To determine whether additional related genes are located in the vicinity of apoAI and apoCIII genes we cloned and extensively characterized approximately 30 kilobases (kb) of human genomic DNA containing these genes and their flanking sequences. Hybridization studies indicated that a non-repetitive DNA fragment located 12kb 3′ to ApoAI gene contains sequences homologous to a 1.8kb mRNA species in human fetal intestine and adult liver but not in fetal liver, kidney, heart, brain or muscle. A cDNA clone was isolated from an adult human liver library by hybridization homology to DNA sequences located 12kb 3′ to apoAI gene. The nucleotide sequences of this cDNA clone is 74.8% homologous to rat apolipoprotein AIV (apoAIV) cDNA and codes for a protein that is 58.6% identical to the rat apoAIV amino acid sequence. These results indicate that apoAI, apoCIII and apoAIV genes are closely linked in the human genome and suggest that all three of them derived from a common ancestral precursor.

The Molecular Basis of the Defect in Familial Combined Apolipoproteins AI and CIII Deficiency

A key feature in atherosclerosis is the progressive accumulation of cholesterol in cells of the arterial wall. Animal cells control their cholesterol content through the integration of the pathways involved in cellular biosynthesis, uptake and secretion of cholesterol. Secreted cholesterol is transported through the plasma to the liver via a series of reactions collectively termed reverse cholesterol transport. A key step in reverse cholesterol is the efflux of cholesterol from cell membranes to a species of high density lipoproteins (HDL). The major protein constituent of HDL is apolipoprotein AI (apoAI). ApoAI activates lecithin: cholesterol acetyltransferse (LCAT), an enzyme involved in esterification of cholesterol on HDL particles. This esterification reaction is thought to be essential for net transport of cholesterol from cells into HDL (reviewed in refs. 1 and 2). These considerations, taken together, imply that deficiency or chronic reduction in plasma HDL and apoAI levels may result in increased risk for atherosclerosis. In support of this possibility, a large number of epidemiological studies have revealed a strong inverse correlation between plasma HDL and apoAI levels and atherosclerosis (reviewed in ref. 3). However, until recently, direct evidence for a cause and effect relationship between plasma HDL/apoAI levels and risk for atherosclerosis was not available. An approach to establish such a relationship is to show that at least in some cases, the only risk factor associated with atherosclerosis is primary (i.e. genetic) HDL deficiency.