APOE Promoter Research Articles

Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes

Cyclooxygenase-2 (COX-2) is upregulated in many cancers, and the prostanoids synthesized increase proliferation, improve angiogenesis, and inhibit apoptosis in several tissues. To explore the function of COX-2 in liver, transgenic (Tg) mice were generated containing a fusion gene (LIVhCOX-2) consisting of human COX-2 cDNA under the control of the human ApoE promoter. Six lines were developed; all of them expressed the LIVhCOX-2 transgene selectively in hepatocytes. The Tg mice exhibited a normal phenotype, and the increased levels of PGE2 found were due to the constitutively expressed COX-2. Histological analysis of different tissues and macroscopic examination of the liver showed no differences between wild-type (Wt) and Tg animals. However, Tg animals were resistant to Fas-mediated liver injury, as demonstrated by low levels of plasmatic aminotransferases, a lesser caspase-3 activation, and Bax levels and an increase in Bcl-2, Mcl-1, and xIAP proteins, when compared with the Wt animals. Moreover, the resistance to Fas-mediated apoptosis is suppressed in the presence of COX-2-selective inhibitors, which prevented prostaglandin accumulation in the liver of Tg mice. These results demonstrate that expression of COX-2-dependent prostaglandins exerted a protection against liver apoptosis.

Liver X Receptor Inhibits the Synthesis and Secretion of Apolipoprotein A1 by Human Liver-Derived Cells

The liver X receptor (LXR) agonist TO901317 inhibited the synthesis of apolipoprotein A1 (apo A1) by human liver-derived cells, including the formation of lipid-poor, prebeta-migrating high-density lipoprotein (HDL). Despite activation of the lipid transporter ABCA1 under these conditions, cellular efflux of PL and cholesterol from liver cells was also reduced. By assaying transcription from full-length and truncated promoters and by site-directed mutagenesis, the effect of LXR and its ligand was localized to a binding site for hepatic nuclear factor-4 (HNF4) in the proximal apo A1 promoter (-132/-119 bp). Chromatin immunoprecipitation analysis of apo A1 transcription complexes from control and ligand-activated cells showed an increase in the binding of reported apo A1 transcriptional inhibitor COUP-TF, which competes with HNF4 for DNA binding. It also identified LXR in the apo A1 transcription complex of TO901317-treated cells. Displacement of HNF4 from the -132/-119 bp promoter DNA sequence in the presence of TO901317 was confirmed by gel shift analysis. These data indicate that LXR can be a significant negative regulator of apo A1 transcription and HDL synthesis.

ABCA1 Overexpression in the Liver of LDLr-KO Mice Leads to Accumulation of Pro-atherogenic Lipoproteins and Enhanced Atherosclerosis

The identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared with LDLr-KO mice, ABCA1-Tg x LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly because of a 2.8-fold increase in cholesterol associated with a large pool of apoB-lipoproteins. ApoB synthesis was unchanged but the catabolism of (125)I-apoB-VLDL and -LDL were significantly delayed, accounting for the 1.35-fold increase in plasma apoB levels in ABCA1-Tg mice. We also found rapid in vivo transfer of free cholesterol from HDL to apoB-lipoproteins in ABCA1-Tg mice, associated with a significant 2.7-fold increase in the LCAT-derived cholesteryl linoleate content found primarily in apoB-lipoproteins. ABCA1-Tg mice had 1.4-fold increased hepatic cholesterol concentrations, leading to a compensatory 71% decrease in de novo hepatic cholesterol synthesis, as well as enhanced biliary cholesterol, and bile acid secretion. CAV-1, CYP2b10, and ABCG1 were significantly induced in ABCA1-overexpressing livers; however, no differences were observed in the hepatic expression of CYP7alpha1, CYP27alpha1, or ABCG5/G8 between ABCA1-Tg and control mice. As expected from the pro-atherogenic plasma lipid profile, aortic atherosclerosis was increased 10-fold in ABCA1-Tg mice. In summary, hepatic overexpression of ABCA1 in LDLr-KO mice leads to: 1) expansion of the pro-atherogenic apoB-lipoprotein cholesterol pool size via enhanced transfer of HDL-cholesterol to apoB-lipoproteins and delayed catabolism of cholesterol-enriched apoB-lipoproteins; 2) increased cholesterol concentration in the liver, resulting in up-regulated hepatobiliary sterol secretion; and 3) significantly enhanced aortic atherosclerotic lesions.

Sustained phenotypic correction in a mouse model of hypoalphalipoproteinemia with a helper-dependent adenovirus vector

We examined the efficacy and host response to the adenovirus (Ad)-mediated delivery of human apolipoprotein A-I (APOA1) gene to the liver of APOA1(-/-) mice. Administration of a first-generation vector (FGAd-AI) resulted in a transient appearance of APOA1 in plasma and induced an anti-APOA1 antibody titer, whereas treatment with a helper-dependent vector (HDAd-AI) resulted in sustained APOA1 expression without inducing an antibody titer. With these results, we studied the effects of FGAd vectors on APOAI expression by HDAd-AI vector. Co-treatment with an FGAd vector inhibited HDAd-AI- mediated APOA1 expression independent of transgene cassettes, but only FGAd-AI induced a humoral response. Furthermore, APOA1 mRNA levels in mice co-treated with FGAd vectors were much lower than those expected from the vector copy number, suggesting that DNA of FGAd vectors interferes with the HDAd-AI vector's APOA1 promoter. A single treatment with an HDAd-AI vector produced a supraphysiological plasma APOA1 level that gradually declined to about half the normal human level over the course of 2 years, associated with a plasma cholesterol level that is persistently higher than that in controls. This investigation provides the proof of principle that liver-directed HDAd gene delivery is effective for the long-term phenotypic correction of monogenic hypoalphalipoproteinemia.

P3-207: Association of the -491 but not -219 APOE promoter polymorphism with Aβ 40 load as CAA in AD

P3-207: Association of the -491 but not -219 APOE promoter polymorphism with Aβ 40 load as CAA in AD

Apolipoprotein AV does not contribute to hypertriglyceridaemia or triglyceride lowering by dietary fish oil and rosiglitazone in obese Zucker rats

Apolipoprotein AV (apoAV) is a recently discovered apolipoprotein with a triglyceride-lowering effect in genetically modified mice. Transcription of the human gene encoding apoAV (APOA5) is suppressed by insulin and stimulated by fibrates. Our goal was to study the expression of Apoa5, in comparison with Apoa4 and Apoc3, in hypertriglyceridaemic, obese and insulin-resistant Zucker rats receiving the insulin sensitiser rosiglitazone and/or a fish oil diet to lower triglycerides. Hepatic Apoa5, Apoa4 and Apo3 mRNA and liver and plasma apoAV were measured in lean and obese Zucker rats receiving rosiglitazone while on a coconut oil or fish oil diet. Basal hepatic Apoa5 expression was similar in obese and lean Zucker rats. Unexpectedly, obese Zucker rats tended to have higher plasma apoAV levels despite their hypertriglyceridaemic state. Both rosiglitazone and the fish oil diet significantly increased Apoa5 mRNA, by about 70%, but tended to lower liver and plasma apoAV. Rosiglitazone had no effect on Apoa5 mRNA in cultured rat hepatocytes. No intact PPAR (peroxisome proliferator-activated receptor) response element was identified in the rat Apoa5 promoter. Our data indicate that apoAV does not contribute to the hypertriglyceridaemia of obese Zucker rats or to the hypolipidaemic effect of rosiglitazone or a fish oil diet. The divergent changes of Apoa5 mRNA and apoAV levels suggest co- or post-translational regulation. The increase in Apoa5 mRNA induced by rosiglitazone is not directly mediated by peroxisome proliferator-activated receptor gamma.

ApoA-I

See related article, pages 564–571 A link between homocysteine and atherothrombotic vascular disease was first suggested by McCully more than 30 years ago.1 The vascular lesions observed by McCully in children with inborn errors of homocysteine metabolism were fibrotic rather than lipid rich, suggesting the possibility that hyperhomocysteinemia may produce vascular pathology through a mechanism independent of altered lipid metabolism. Since that time, many epidemiological studies have confirmed that elevated plasma levels of total homocysteine (tHcy) are associated with an increased risk of coronary events, stroke, and venous thromboembolism.2–4 Most of the epidemiological data indicate that elevation of plasma tHcy is not associated with a significant change in plasma total cholesterol but is negatively associated with high density lipoprotein (HDL) cholesterol.5–7 An effect of homocysteine on HDL metabolism could be clinically important, because HDL protects from vascular disease not only by facilitating reverse cholesterol transport8 but also through its direct antiinflammatory properties.9 During the past decade, the development of animal models has led to rapid progress in defining the pathophysiological consequences of hyperhomocysteinemia in vivo. Experimental approaches to induce hyperhomocysteinemia in animals include the use of diets that are high in methionine and/or low in folate, as well as genetic approaches, such as the generation of knockout mice with targeted disruption of the cystathionine β-synthase ( Cbs ),10 methylene tetrahydrofolate reductase ( Mthfr ),11 or methionine synthase ( Mtr )12 genes. Like hyperhomocysteinemic humans, hyperhomocysteinemic animals develop endothelial dysfunction, hepatic lipid accumulation, and decreased plasma HDL cholesterol with minimal changes in plasma …

Inhibition of Apolipoprotein AI Gene Expression by Tumor Necrosis Factor α: Roles for MEK/ERK and JNK Signaling

Plasma high-density lipoprotein and apolipoprotein AI (apoAI) levels are suppressed by tumor necrosis factor alpha. To determine the molecular mechanisms responsible for the effect of TNF alpha on the apoAI promoter activity, HepG2 cells were exposed to both genetic and pharmacological modulators of TNF alpha-mediated signaling in the presence or absence of TNF alpha. Exogenous ERK1 and ERK2 expression suppressed basal apoAI promoter activity; however, only ERK2 enhanced the ability of TNF alpha to suppress apoAI promoter activity. Exogenous expression of all three MEK isoforms (MEK1, MEK2A, and MEK2E) suppressed basal apoAI promoter activity and further aggravated TNF alpha-related apoAI promoter activity inhibition. Treatment with SB202190 (p38 MAP kinase inhibitor) alone significantly increased apoAI promoter activity; however, in the presence of TNF alpha, apoAI promoter activity was suppressed to an extent similar to that in cells not treated with SB202190. ApoAI promoter activity increased in cells treated with the specific JNK inhibitor SP600125, but unlike SB202190 treatment, the level of TNF alpha-related apoAI promoter inhibition was reduced by 50%. Similarly, the level of TNF alpha-related apoAI promoter inhibition was reduced in cells transfected with JNK1 siRNA. Finally, treatment of cells with the NF-kappaB inhibitors BAY and SN-50 or overexpression of NF-kappaB subunits p50 and p65 had no effect on the ability of TNF alpha to repress apoAI promoter activity. These results suggest that TNF alpha suppresses apoAI promoter activity through both the MEK/ERK and JNK pathways but is not mediated by either p38 MAP kinase activity or NF-kappaB activation.

Elevated Homocysteine Reduces Apolipoprotein A-I Expression in Hyperhomocysteinemic Mice and in Males With Coronary Artery Disease

Hyperhomocysteinemia, a risk factor for cardiovascular disease, is caused by nutritional or genetic disturbances in homocysteine metabolism. A polymorphism in methylenetetrahydrofolate reductase (MTHFR) is the most common genetic cause of mild hyperhomocysteinemia. To examine mechanisms by which an elevation in plasma homocysteine leads to vascular disease, we first performed microarray analyses in livers of Mthfr-deficient mice and identified differentially expressed genes that are involved in lipid and cholesterol metabolism. Microarrays and RT-PCR showed decreased mRNA for apolipoprotein A (ApoA)-IV and for ApoA-I and increased mRNA for cholesterol 7alpha hydroxylase (Cyp7A1) in Mthfr(+/-) mice compared with Mthfr(+/+) mice. Western blotting revealed that ApoA-I protein levels in liver and plasma of Mthfr(+/-) mice were 52% and 62% of levels in the respective tissues of Mthfr(+/+) mice. We also performed Western analysis for plasma ApoA-I protein levels in 60 males with coronary artery disease and identified a significant (P<0.01) negative correlation (-0.33) between ApoA-I and plasma homocysteine levels. This cohort also displayed a negative correlation (-0.24, P=0.06) between high-density lipoprotein cholesterol and plasma homocysteine. Treatment of HepG2 cells with supraphysiological levels of 5 mmol/L homocysteine reduced peroxisome proliferator-activated receptor (PPAR) alpha and ApoA-I protein levels and decreased ApoA-I promoter activity. Transfection with a PPARalpha construct upregulated ApoA-I and MTHFR. Our results suggest that hyperhomocysteinemia may increase risk of atherosclerosis by decreasing expression of ApoA-I and increasing expression of CYP7A1.

Ascorbic acid and α-tocopherol down-regulate apolipoprotein A-I gene expression in HepG2 and Caco-2 cell lines

HepG2 cells and Caco-2 cells were treated with various concentrations of select antioxidants to study some of the molecular pathways underlying antioxidant-related changes in apolipoprotein A-I (apoA-I) expression. Both α-tocopherol and ascorbate treatment over a time course of 72 hours caused a significant reduction in apoA-I messenger RNA and protein levels in a dose-dependent fashion. Albumin levels did not change with any treatment, suggesting that the effect is protein-specific. Similar changes were seen in Caco-2 cells. In contrast, apoA-I messenger RNA and protein levels significantly increased after 28 and 280 μmol/L dimethyl sulfoxide (DMSO) treatment. Cells were transfected with chloramphenicol acetyltransferase (CAT) reporter gene plasmid containing the full-length apoA-I promoter to determine if these changes occur at a transcriptional level, and after 24 hours, the HepG2 or Caco-2 cells were treated with varying concentrations of ascorbate or α-tocopherol. At the highest concentration of ascorbate and α-tocopherol used (5 mmol/L), there was a significant reduction in apoA-I promoter activity. DMSO treatment up-regulated apoA-I promoter activity, whereas increasing oxidative load with 50, 100, and 200 μmol/L hydrogen peroxide treatment did not significantly alter apoA-I promoter activity. Studies with deletional constructs of the promoter containing or lacking the antioxidant response element (ARE) indicated that the effect of ascorbate and α-tocopherol, unlike that of DMSO, was independent of this ARE. Using a series of apoA-I deletion constructs, it was shown that site A–containing segment of the promoter has a critical regulatory element. However, electromobility shift assays indicated that there was no significant change in nuclear factor binding activity to site A as a result of treatment with ascorbate or α-tocopherol. As expected, treatment with DMSO increased factor binding to the previously described ARE. It is concluded that the apoA-I promoter-stimulating effect of DMSO may be independent of its antioxidant activity and that some antioxidants at very high concentrations may have suppressive effect on the apoA-I gene expression. It appears that the inhibitory effect of ascorbate or α-tocopherol on the apoA-I promoter is either indirect or is the result of posttranslational modifications of the nuclear binding factors. The previously described ARE is not a response element for the ascorbate or α-tocopherol.

Inhibition of apolipoprotein AI gene expression by 1, 25-dihydroxyvitamin D 3

Members of the steroid receptor superfamily are known to alter the transcription of apolipoprotein AI (apo AI), the major apoprotein of high-density lipoprotein (HDL). To assess the role of vitamin D receptor (VDR) in apo AI gene expression, we investigated the effect of 1α, 25-dihydroxycholecalciferol (1, 25-(OH) 2 D 3) as well as the vitamin D antagonist ZK-191784 (ZK), on apo AI gene expression and promoter activity in the human hepatoma cell line HepG2. Apo AI secretion and mRNA levels were both suppressed in a dose-dependent manner in HepG2 cells treated 1, 25-(OH) 2 D 3. This was accompanied by a similar decrease in apo AI promoter activity. Mapping of the vitamin D response element showed that suppression required a region of the apo AI gene promoter identified previously to contain site A. However, vitamin D treatment had no effect on nuclear factor binding to site A of the apo AI promoter. Treatment with vitamin D receptor antagonist ZK inhibited the ability of 1, 25-(OH) 2 D 3 to repress apo AI promoter activity, while higher doses of ZK increased apo AI promoter activity. ZK did not alter estradiol stimulated apo AI promoter activity. The VDR antisense ODN had no effect on apo AI promoter activity in control cells, however, it reversed the repression normally seen in cells treated with 1, 25-(OH) 2D 3. It is concluded that 1, 25-(OH) 2 D 3 suppresses apo A1 gene expression at the transcriptional level, possibly by altering coactivators or corepressors. This effect requires the VDR as well as a vitamin D response element in the apo AI promoter.

APOE Gene and APOE Promoter Polymorphism G-219T Are Not Related to Neuropsychological Test Score in College Football and Soccer Players

APOE Gene and APOE Promoter Polymorphism G-219T Are Not Related to Neuropsychological Test Score in College Football and Soccer Players

Cholesterol-Alzheimer's Link Backed by New Data

Back to table of contents Previous article Next article Clinical & Research NewsFull AccessCholesterol-Alzheimer's Link Backed by New DataJoan Arehart-TreichelJoan Arehart-TreichelSearch for more papers by this authorPublished Online:19 Aug 2005https://doi.org/10.1176/pn.40.16.00400017The e4 variant of the APOE gene has been strongly linked with susceptibility to Alzheimer's disease. Indeed, one-fourth of the population is estimated to carry this variant. What fewer people are aware of, however, is that the APOE gene is also known to be the major transporter of cholesterol in the blood and central nervous system and a risk factor for coronary disease.Now a number of other genes involved in cholesterol metabolism appear to be involved in Alzheimer's susceptibility as well, a new study suggests. However, the APOE gene still seems to be the biggest culprit.The study was led by Andreas Papassotiropoulos, M.D., a psychiatrist and research professor at the University of Zurich in Switzerland. Results appeared in the July Journal of Clinical Psychiatry.Papassotiropoulos and his colleagues enrolled more than 500 older persons from Switzerland and Greece. Half had been diagnosed with Alzheimer's disease; the other half did not have the illness. The researchers then analyzed DNA samples from these subjects to assess 12 single nucleotide polymorphisms—that is, variants in genetic material—from 11 genes related to cholesterol metabolism and previously linked to Alzheimer's disease. One of the single nucleotide polymorphisms was the APOE e4 variant.The scientists also examined the DNA samples to see whether they contained 48 single nucleotide polymorphisms that had never been linked with either cholesterol or Alzheimer's.Finally, they looked for links between any of the single nucleotide polymorphisms and the presence of Alzheimer's.They found that nine of the 12 single nucleotide polymorphisms previously implicated in cholesterol metabolism and Alzheimer's were also significantly associated with Alzheimer's in their study.In contrast, they were not able to find any association between Alzheimer's and the 48 single nucleotide polymorphisms not previously tied to cholesterol and Alzheimer's.Moreover, they found that some of the nine single nucleotide polymorphisms were more strongly associated with Alzheimer's than the others were. The APOE e4 gene variant headed the list. Then came material from a gene called SOAT1. Then came material from the APOE promoter.“This study is potentially important for several reasons,” Eric Reiman, M.D., a professor of psychiatry at the University of Arizona and deputy editor of the Journal of Clinical Psychiatry, asserted in an editorial accompanying the report. For example, “It implicates a cluster of genes, including, but not limited to, the APOE e4 allele, in the susceptibility to late-onset Alzheimer's disease.”Also, he added, “It provides further support for the role of higher cholesterol levels in the pathogenesis of Alzheimer's disease and the potential role of cholesterol-lowering strategies in the treatment and prevention of this disorder.”However, as Reiman cautioned in an interview with Psychiatric News, the findings will not be used clinically any time soon to predict Alzheimer's for several reasons—they have not yet been replicated,“ they do not predict with sufficient certainty whether or when a person might develop symptoms,” and “they don't yet tell the person what he or she might be able to do to prevent the disorder.”In fact, he said, testing for the APOE e4 variant alone to predict Alzheimer's risk is not recommended for clinical use at this point because of the danger of triggering false alarms or creating false reassurances, and because no preventive measures are available.Josepha Cheong, M.D., an associate professor of psychiatry at the University of Florida and chair of the APA Council on Aging, told Psychiatric News that the study impressed her for several reasons. For one, the researchers attempted to identify the interplay of a cluster of genes that may be involved in the etiology and clinical presentation of Alzheimer's disease instead of looking for the effect of a single gene at a time, which is more common. For another, she believes that the study“ represents a continuation in the discovery of genes involved in the pathogenesis of Alzheimer's disease.” And finally, whereas the“ research results may not be acutely clinically relevant, they portend significant possibilities for diagnosis and treatment.”Meanwhile, Papassotiropoulos said, “I anticipate that several genetics groups will attempt to replicate the findings, among others Dr. Jonathan Prince from the Karolinska Institute in Sweden. There are [also] several [other] lipid-related genes to be looked at. In addition, it will be important to combine this genetic information with data on brain activation patterns by means of PET or fMRI.”The study was funded by the Swiss National Science Foundation, the Early Diagnosis of Alzheimer's Disease and Related Dementia Program in Switzerland, and the National Center for Competence in Research in Switzerland.“A Cluster of Cholesterol-Related Genes Confers Susceptibility for Alzheimer's Disease” can be accessed online at<www.psychiatrist.com/toc.htm> by searching on the article title in the July issue. ▪ ISSUES NewArchived

Thyroid Hormone Regulates the Hypotriglyceridemic Gene APOA5

The apolipoprotein AV gene (APOA5) is a key determinant of plasma triglyceride levels, a major risk factor for coronary artery disease and a biomarker for the metabolic syndrome. Since thyroid hormones influence very low density lipoprotein triglyceride metabolism and clinical studies have demonstrated an inverse correlation between thyroid status and plasma triglyceride levels, we examined whether APOA5 is regulated by thyroid hormone. Here we report that 3,5,3'-triiodo-L-thyronine (T3) and a synthetic thyroid receptor beta (TRbeta) ligand increase APOA5 mRNA and protein levels in hepatocytes. Our data revealed that T3-activated TR directly regulates APOA5 promoter through a functional direct repeat separated by four nucleotides (DR4). Interestingly, we show that upstream stimulatory factor 1, a transcription factor associated with familial combined hyperlipidemia and elevated triglyceride levels in humans, and upstream stimulatory factor 2 cooperate with TR, resulting in a synergistic activation of APOA5 promoter in a ligand-dependent manner via an adjacent E-box motif. In rats, we observed that apoAV levels declines with thyroid hormone depletion but returned to normal levels upon T3 administration. In addition, treatments with a TRbeta-selective agonist increased apoAV and diminished triglyceride levels. The identification of APOA5 as a T3 target gene provides a new potential mechanism whereby thyroid hormones can influence triglyceride homeostasis. Additionally, these data suggest that TRbeta may be a potential pharmacological target for the treatment of hypertriglyceridemia.

Is there a relation between APOE expression and brain amyloid load in Alzheimer’s disease?

Background: It has been proposed that, independent of the ε4 allele, APOE promoter polymorphisms (−491 A/T and −219 G/T) may be risks factor for Alzheimer’s disease by modulating APOE expression....

ApoE −491A/T Promoter Polymorphism is not an Independent Risk Factor, but Associated with the ε4 Allele in Hungarian Alzheimer’s Dementia Population

Apolipoprotein E gene (Apo(epsilon)) has three common alleles (epsilon2, epsilon3, and epsilon4), of which epsilon4 has been shown to be associated with an increased risk for Alzheimer's disease (AD). Possible additional genetic factors, like the -491A variant of ApoE promoter may modify the development of AD, independently of the ApoE allele status. The objective of this study was to investigate whether A/T allelic polymorphism at site-491 of the ApoE promoter is associated with AD in a Hungarian population. The genomic DNA isolated from peripheral blood lymphocytes of 52 late-onset AD and 53 control individuals was used as a template for the two examined polymorphisms and PCR assay was applied. The epsilon4 allele was significantly over-represented in the AD group (28%) as compared with the control population (7%). No significant differences have been found between the control and the AD populations regarding the occurrence of the promoter A allele frequencies (control: 77%, AD: 70%). However, the AA genotype was more frequent in the AD group (48%) than in the control (10%) when the presence of epsilon4 allele was also considered. It is unlikely therefore that the -491A variant of the ApoE promoter gene is an independent risk factor in the Hungarian AD population, but a linkage disequilibrium exists between the two examined mutations.

A conserved retinoid X receptor (RXR) from the mollusk Biomphalaria glabrata transactivates transcription in the presence of retinoids

Retinoid X receptors (RXR) are members of the nuclear receptor superfamily of ligand-activated transcription factors that have been characterized in a wide variety of metazoan phyla. They act as heterodimer partners of other nuclear receptors, and in vertebrates also activate transcription as homodimers in the presence of a ligand, 9-cis retinoic acid. In order to test the hypothesis that retinoic acid signaling pathways involving RXRs are present in the Lophotrochozoa, we have sought to isolate conserved members of this family from the platyhelminth parasite Schistosoma mansoni and its intermediate host, the mollusk Biomphalaria glabrata. Here we report that an RXR ortholog from B. glabrata (BgRXR) is better conserved, compared with mouse RXRalpha, both in the DNA-binding domain (89% identity) and in the ligand-binding domain (LBD) (81% identity), than are arthropod homologs. In EMSA, BgRXR binds to the direct repeat response element DR1 as a homodimer or as a heterodimer with mammalian RARalpha, LXR, FXR or PPARalpha. When transfected alone into mammalian cell lines, BgRXR transactivated transcription of a reporter gene from the Apo-A1 promoter in the presence of 9-cis retinoic acid or DHA. Constructs with the Gal4 DNA binding domain fused to the hinge and LBDs of BgRXR were used to show that ligand-dependent activation of transcription by BgRXR required its intact AF-2 activation domain, and that the LBD can form homodimers. Finally, the binding of 9-cis retinoic acid preferentially protected the LBD of BgRXR from degradation by trypsin in a proteolysis protection assay. Our results show that BgRXR binds and is activated by retinoids and suggest that retinoid signaling pathways are conserved in the Lophotrochozoa. The nucleotide sequence reported in this paper has been submitted to the GenBank/EBI Data Bank with accession no. AY048663.

Transgenic mice overexpressing murine thrombopoietin develop myelofibrosis and osteosclerosis

Thrombopoietin (TPO) regulates megakaryocytopoiesis and platelet production in vivo and in vitro. Exogenous overexpression of TPO in vivo by viral-mediated gene transfer induced bone marrow (BM) fibrosis and osteosclerosis. On the other hand, transgenic mice (Tg) overexpressing TPO using a liver-specific apolipoprotein E (Apo-E) promoter did not exhibit myelofibrosis or osteosclerosis. These discrepancies in phenotype are not fully understood. Then we have investigated the consequences of long-term in vivo overexpression of TPO in a mouse model. Murine TPO Tg mice driven by the IgH promoter were generated. The number of platelets and neutrophils in peripheral blood, and the number of megakaryocytes and granulocytic immature cells in the BM was elevated, together with the number of progenitor cells for megakaryocyte and myeloid cells. TPO Tg mice demonstrated anemia but the number of progenitor cells for the erythrocyte was increased. TPO Tg mice developed myelofibrosis and osteosclerosis as they aged with extramedullary hematopoiesis in the spleen. As plasma transforming growth factors (TGF)-β1 and osteoprotegerin (OPG) levels were higher in TPO Tg mice than in wild-type mice, the development of myelofibrosis and osteosclerosis depends on local TPO levels in BM and might be due to elevated TGF-β1 and OPG.

Temporal Control of Cre Recombinase-mediated in Vitro DNA Recombination by Tet-on Gene Expression System

Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in mouse models. These strategies exploiting Cre-mediated site-specific DNA recombination have been incorporated into transgenic and gene-targeting procedures to allow in vivo manipulation of DNA in embryonic stem cells (ES cells) or living animals. The Cre/lox P system has become widely used in conditional gene targeting, conditional gene repair and activation, inducible chromosome translocation, and chromosome engineering. In this project, we have employed the universal transgenic system and the liver-specific promoter system for tightly temporal and liver-specific control of Cre gene expression in mice that (1) integrates the advantages of the Tet-on gene expression system and Cre/lox P site-mediated gene activation, and (2) simplifies the scheme of animal crosses through a combination of two control elements in a single transgene. A liver-specific apoE promoter was inserted into the promoter cloning site upstream of the rtTA cassette of pCore construct to generate the transgene construct pApoErtTA-tetO-Cre, followed by demonstrating stringent regulation of doxycycline (Dox)-induced Cre-mediated recombination in the lox P-flanked transcription STOP cassette-modified BEL-7402 cells. That is to say, in the absence of Dox, the Cre gene is not expressed and will not induce site-specific recombination between two lox P sites, whereas on exposure to Dox, the Cre gene will be expressed and the recombination will occur. Together, these data indicate that the Tet-on gene expression system is able to successfully and stringently control Cre expression in vitro, which lays a solid foundation for efficient and spatio-temporal Cre gene activation in transgenic mice.

Temporal Control of Cre Recombinase-mediated &amp;lt;italic&amp;gt;in Vitro&amp;lt;/italic&amp;gt; DNA Recombination by Tet-on Gene Expression System

Abstract Conditional gene expression and gene deletion are important experimental approaches for examining the functions of particular gene products in mouse models. These strategies exploiting Cre-mediated site-specific DNA recombination have been incorporated into transgenic and gene-targeting procedures to allow in vivo manipulation of DNA in embryonic stem cells (ES cells) or living animals. The Cre/lox P system has become widely used in conditional gene targeting, conditional gene repair and activation, inducible chromosome translocation, and chromosome engineering. In this project, we have employed the universal transgenic system and the liver-specific promoter system for tightly temporal and liver-specific control of Cre gene expression in mice that (1) integrates the advantages of the Tet-on gene expression system and Cre/lox P site-mediated gene activation, and (2) simplifies the scheme of animal crosses through a combination of two control elements in a single transgene. A liver-specific apoE promoter was inserted into the promoter cloning site upstream of the rtTA cassette of pCore construct to generate the transgene construct pApoErtTA-tetO-Cre, followed by demonstrating stringent regulation of doxycycline (Dox)-induced Cre-mediated recombination in the lox P-flanked transcription STOP cassette-modified BEL-7402 cells. That is to say, in the absence of Dox, the Cre gene is not expressed and will not induce site-specific recombination between two lox P sites, whereas on exposure to Dox, the Cre gene will be expressed and the recombination will occur. Together, these data indicate that the Tet-on gene expression system is able to successfully and stringently control Cre expression in vitro, which lays a solid foundation for efficient and spatio-temporal Cre gene activation in transgenic mice.