Low Density Lipoprotein Receptor Promoter Research Articles

Flavonoids regulate LDLR through different mechanisms tied to their specific structures

Flavonoids, polyphenolic compounds found in plant-based diets, are associated with reduced risk of cardiovascular disease and longevity. These components are reported to reduce plasma levels of low-density lipoprotein (LDL) through an upregulation of the LDL receptor (LDLR), but the mechanism is still largely unknown. In this study, we have systematically screened the effect of 12 flavonoids from six different flavonoid subclasses on the effect on LDLR. This paper provides an in-depth analysis on how these flavonoids affect LDLR regulation and functionality. We found that most but not all of the tested flavonoids increased LDLR mRNA levels. Surprisingly, this increase was attributed to different regulatory mechanisms, such as enhanced LDLR promoter activity, LDLR mRNA stabilization, or LDLR protein stabilization, of which specific effectual parts of the flavonoid molecular structure could be assigned. These types of comparative analysis of various flavonoids enhance clarity and deepen the understanding of how the different structures of flavonoids affect LDLR regulation. Our data offer useful insights that may guide future research in developing therapeutic approaches for cardiovascular health.

Chrysin 7-O-β-d-glucopyranoside increases hepatic low-density lipoprotein receptor expression through AMP-activated protein kinase activation.

Elevated plasma low-density lipoprotein (LDL) cholesterol level is a risk factor for developing atherosclerosis. Increased LDL receptor (LDLR) expression is expected to reduce the risk of atherosclerotic disease since hepatic LDLR is essential for clearing plasma LDL cholesterol. Here, we screened human LDLR promoter effectors and observed that extracts from peduncles of sweet cherry (Prunus avium) 'Sato-Nishiki' induce LDLR gene promoter activity. We used several analytical and chemical methods to show that chrysin 7-O-β-d-glucopyranoside (chrysin-7G) is one of the compounds that stimulate LDLR gene promoter activity in cherry peduncle extracts. Furthermore, synthetic chrysin-7G increased the expression and activity of LDLR. The chrysin-7G-mediated increase in LDLR expression and activity was completely abolished by treatment with an AMP-activated protein kinase (AMPK) inhibitor, compound C. These results indicate that chrysin-7G increases LDLR expression through AMPK activation and may be a useful compound that can be recycled from waste parts of agricultural products.

HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation.

[Figure: see text].

Transcription Factor MAFF (MAF Basic Leucine Zipper Transcription Factor F) Regulates an Atherosclerosis Relevant Network Connecting Inflammation and Cholesterol Metabolism.

Coronary artery disease (CAD) is a multifactorial condition with both genetic and exogenous causes. The contribution of tissue-specific functional networks to the development of atherosclerosis remains largely unclear. The aim of this study was to identify and characterize central regulators and networks leading to atherosclerosis. Based on several hundred genes known to affect atherosclerosis risk in mouse (as demonstrated in knockout models) and human (as shown by genome-wide association studies), liver gene regulatory networks were modeled. The hierarchical order and regulatory directions of genes within the network were based on Bayesian prediction models, as well as experimental studies including chromatin immunoprecipitation DNA-sequencing, chromatin immunoprecipitation mass spectrometry, overexpression, small interfering RNA knockdown in mouse and human liver cells, and knockout mouse experiments. Bioinformatics and correlation analyses were used to clarify associations between central genes and CAD phenotypes in both human and mouse. The transcription factor MAFF (MAF basic leucine zipper transcription factor F) interacted as a key driver of a liver network with 3 human genes at CAD genome-wide association studies loci and 11 atherosclerotic murine genes. Most importantly, expression levels of the low-density lipoprotein receptor (LDLR) gene correlated with MAFF in 600 CAD patients undergoing bypass surgery (STARNET [Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task]) and a hybrid mouse diversity panel involving 105 different inbred mouse strains. Molecular mechanisms of MAFF were tested in noninflammatory conditions and showed positive correlation between MAFF and LDLR in vitro and in vivo. Interestingly, after lipopolysaccharide stimulation (inflammatory conditions), an inverse correlation between MAFF and LDLR in vitro and in vivo was observed. Chromatin immunoprecipitation mass spectrometry revealed that the human CAD genome-wide association studies candidate BACH1 (BTB domain and CNC homolog 1) assists MAFF in the presence of lipopolysaccharide stimulation with respective heterodimers binding at the MAF recognition element of the LDLR promoter to transcriptionally downregulate LDLR expression. The transcription factor MAFF was identified as a novel central regulator of an atherosclerosis/CAD-relevant liver network. MAFF triggered context-specific expression of LDLR and other genes known to affect CAD risk. Our results suggest that MAFF is a missing link between inflammation, lipid and lipoprotein metabolism, and a possible treatment target.

23,24-Dihydrocucurbitacin B promotes lipid clearance by dual transcriptional regulation of LDLR and PCSK9.

23,24-Dihydrocucurbitacin B (designated as C95 in this article) is a cucurbitane triterpenoid that has been shown to possess a variety of pharmacological activities, such as anti-inflammatory and anti-HIV-1 activities etc. In this study, we investigated the effects of 23,24-dihydrocucurbitacin B on lipid regulation. We showed that 23,24-dihydrocucurbitacin B (1–5 μM) dose-dependently promoted DiI-LDL uptake in HepG2 cells by upregulating low-density lipoprotein receptor (LDLR) protein. In HepG2 cells, 23,24-dihydrocucurbitacin B (1–10 μM) dose-dependently enhanced LDLR promoter activity by elevating the mature form of SREBP2 (sterol regulatory element binding protein 2) protein levels on one hand, and inhibited PCSK9 (proprotein convertase subtilisin/kexin type 9) promoter activity by attenuating HNF1α (hepatocyte nuclear factor-1α) protein levels in nuclei on the other hand. Consequently, the expression of LDLR protein markedly increased, whereas the PCSK9-mediated LDLR protein degradation decreased. In a high-cholesterol LVG golden Syrian Hamster model, administration of 23,24-dihydrocucurbitacin B (30 mg · kg−1⋅ d−1, intragastric, for 3 weeks) significantly decreased the serum LDL-cholesterol (LDL-C) levels. PCSK9 protein levels in the serum and liver tissues were significantly decreased, whereas LDLR protein levels in liver tissues were significantly increased in the treated animals as compared with the control animals. In conclusion, our study demonstrates for the first time that 23,24-dihydrocucurbitacin B exhibits dual transcriptional regulation of LDLR and PCSK9 in HepG2 cells by increasing SREBP2 protein levels and decreasing HNF1α protein levels in the nuclei. These results propose a new strategy to simultaneously manage LDLR and PCSK9 protein expression and provide a promising lead compound for drug development.

Inhibitors of AKT kinase increase LDL receptor mRNA expression by two different mechanisms.

Protein kinase B (AKT) is a serine/threonine kinase that functions as an important downstream effector of phosphoinositide 3-kinase. We have recently shown that MK-2206 and triciribine, two highly selective AKT inhibitors increase the level of low density lipoprotein receptor (LDLR) mRNA which leads to increased amount of cell-surface LDLRs. However, whereas MK-2206 induces transcription of the LDLR gene, triciribine stabilizes LDLR mRNA, raising the possibility that the two inhibitors may actually affect other kinases than AKT. In this study, we aimed to ascertain the role of AKT in regulation of LDLR mRNA expression by examining the effect of five additional AKT inhibitors on LDLR mRNA levels. Here we show that in cultured HepG2 cells, AKT inhibitors ARQ-092, AKT inhibitor VIII, perifosine, AT7867 and CCT128930 increase LDLR mRNA levels by inducing the activity of LDLR promoter. CCT128930 also increased the stability of LDLR mRNA. To study the role of AKT isoforms on LDLR mRNA levels, we examined the effect of siRNA-mediated knockdown of AKT1 or AKT2 on LDLR promoter activity and LDLR mRNA stability. Whereas knockdown of either AKT1 or AKT2 led to upregulation of LDLR promoter activity, only knockdown of AKT2 had a stabilizing effect on LDLR mRNA. Taken together, these results provide strong evidence for involvement of AKT in regulation of LDLR mRNA expression, and point towards the AKT isoform specificity for upregulation of LDLR mRNA expression.

Evidence of association of circulating epigenetic-sensitive biomarkers with suspected coronary heart disease evaluated by Cardiac Computed Tomography.

Circulating biomarkers available in clinical practice do not allow to stratify patients with coronary heart disease (CHD) prior the onset of a clinically relevant event. We evaluated the methylation status of specific genomic segments and gene expression in peripheral blood of patients undergoing Cardiac Computed Tomography (CCT) for CHD (n = 95).We choose to investigate cholesterol metabolism. Methylation and gene expression of low density lipoprotein receptor (LDLR), sterol regulatory element-binding factor 2 (SREBF2) and ATP-binding cassette transporter 1 (ABCA1) were evaluated by qRT-PCR. Calcium score (CACS), stenosis degree, total plaque volume (TPV), calcified plaque volume (CPV), non-calcified plaque volume (NCPV) and plaque burden (PB) were assessed in all CHD patients (n = 65). The percentage of methylation at the specific analyzed segment of LDLR promoter was higher in CHD patients vs healthy subjects (HS) (n = 30) (p = 0.001). LDLR, SREBF2 and ABCA1 mRNAs were up-regulated in CHD patients vs HS (p = 0.02; p = 0.019; p = 0.008). SREBF2 was overexpressed in patients with coronary stenosis ≥50% vs subjects with stenosis <50% (p = 0.036). After adjustment for risk factors and clinical features, ABCA1 (p = 0.005) and SREBF2 (p = 0.010) gene expression were identified as independent predictors of CHD and severity. ROC curve analysis revealed a good performance of ABCA1 on predicting CHD (AUC = 0.768; p<0.001) and of SREBF2 for the prediction of disease severity (AUC = 0.815; p<0.001). Moreover, adjusted multivariate analysis demonstrated SREBF2 as independent predictor of CPV, NCPV and TPV (p = 0.022; p = 0.002 and p = 0.006) and ABCA1 as independent predictor of NCPV and TPV (p = 0.002 and p = 0.013).CHD presence and characteristics are related to selected circulating transcriptional and epigenetic-sensitive biomarkers linked to cholesterol pathway. More extensive analysis of CHD phenotypes and circulating biomarkers might improve and personalize cardiovascular risk stratification in the clinical settings.

Modulation of LDL receptor expression and promoter methylation in HepG2 cells treated with a Corylus avellana L. extract

The aim of our study was to evaluate the impact of an ethanolic extract of C. avellana on the molecular pathway(s) regulating the low-density lipoprotein receptor (LDLR) in HepG2 cells, mainly in terms of epigenetics. We demonstrated that viability, proliferation and cell cycle distribution were not affected up to 72 h of treatment, whereas LDLR expression was stimulated as early as 24 h following administration (P < 0.05 at 0.04 mg/ml, P < 0.01 at 0.4 mg/ml). The level of DNA Methyl Transferase 3A was up-regulated (P < 0.001 at 0.004 mg/ml, P < 0.05 at 0.4 and 4 mg/ml), without any change in global DNA methylation, whereas the percentage of 5-methyl cytosine was significantly (P < 0.05) reduced at LDLR promoter level in response to treatment (0.04 mg/ml). Overall, our data demonstrate that the ethanolic extract of C. avellana stimulates the LDLR expression in HepG2 cells by epigenetic mechanisms.

Neoagarooligosaccharides enhance the level and efficiency of LDL receptor and improve cholesterol homeostasis

Neoagarooligosaccharides (NAOS) are produced by β-agarases through the cleavage of β-1,4 linkage in agarose. Here, we investigated the cholesterol-lowering properties of NAOS and its underlying molecular mechanism. Administration of NAOS in mice fed a high-cholesterol diet (HCD) inhibited hepatic fat accumulation and serum LDL cholesterol. Mice fed an HCD showed a decrease in LDL receptor (LDLR) expression, which was reversed by NAOS. Moreover, NAOS enhanced LDLR expression and LDL uptake in hepatocytes. NAOS increased nuclear translocation of SREBP-2, a crucial transcription factor for LDLR gene expression. Increased LDLR luciferase activity, induced by NAOS, was abolished on deletion of SRE in the LDLR promoter. NAOS upregulated LDLR levels via PI3K/Akt-mediated activation of SREBP-2. Furthermore, we demonstrated that NAOS reduced the nuclear HNF-1α, leading to decreased levels of PCSK9, a natural inhibitor of LDLR. These findings indicate that NAOS exhibits hypolipidemic activity, and may serve as a new hypolipidemic drug.

Role of hesperetin in LDL-receptor expression in hepatoma HepG2 cells.

BackgroundHigh plasma concentration of low-density lipoprotein cholesterol (LDL-c) plays a significant role in the incidence of atherosclerosis and coronary heart diseases. The aim of this study was to investigate the mechanism by which the citrus flavonoid, hesperetin, regulates the LDL receptor (LDLr) gene in the human liver using the human hepatoma cell line, HepG2.MethodsLuciferase reporter gene assays were performed (in the absence of lipoprotein) to measure the activity of the LDLr promoter and the promoters of the sterol regulatory element binding protein (SREBP) transcription factors that control the LDLr promoter.ResultsOnly SREBP-1 promoter activity was significantly increased 4 h after exposure to 200 μM hesperetin. However, after 24 h incubation with 200 μM hesperetin, the activities of all the promoter-constructs, SREBP-1a, -1c, -2 and LDLr, were significantly increased. The effects of 200 μM hesperetin on elevating LDLr mRNA levels were possibly due to regulation of LDLr gene transcription by SREBP-la and SREBP-2.ConclusionsWe conclude that 200 μM hesperetin was likely to have stimulated LDLr gene expression in human hepatoma HepG2 cells via increased phosphorylation of PI3K andERK1/2, which increased SREBP-1a and SREBP-2 mRNA levels and enhanced the maturation of the encoded proteins. This may lead to lower plasma LDL cholesterol; therefore, diets supplemented with hesperidin might provide cardio-protective effects and reduce mortality and morbidity from coronary heart diseases.

19. Using a Novel Combinatorial Non-Viral Vector and Small Molecules to Treat Familial Hypercholesterolaemia (FH)

Familial hypercholesterolaemia (FH) is a life-threatening genetic disorder characterised by elevated levels of plasma low density lipoprotein cholesterol (LDL-C). Loss-of-function mutations in the gene encoding the low density lipoprotein receptor (LDLR) are responsible for ~85% of all FH cases. We have previously generated mini-gene vectors carrying the human LDLR cDNA, driven by 10 kb of genomic DNA from the native human LDLR locus, encompassing a promoter region with all essential elements required for physiologically regulated expression (pLDLR-LDLR). We demonstrated that incorporation of the genomic DNA promoter elements resulted in long-term physiologically-regulated LDLR transgene expression that complemented Ldlr deficiency. Here we further enhance LDLR transgene expression by characterising and cloning in a miRNA, targeting Hmgcr (miR82) generating a combinatorial RNAi-LDLR vector (pLDLR-LDLR-miR82). We show in vivo that the combinatorial vector efficiently suppresses endogenous Hmgcr transcripts, which leads to an increase in LDLR transgene expression through induction of the LDLR promoter. In a preliminary study the pLDLR-LDLR-miR82 vector was able to significantly reduce total and LDL-C, in Ldlr−/− mice fed a 1% cholesterol diet at two and four weeks post vector delivery. We then carried out a longer term study in Ldlr−/− mice fed a 0.25% cholesterol diet, LDLR expression could be detected 12-weeks post-delivery with the plasmid able to be rescued as a functioning episome. LDL-C was significantly lowered throughout the study and this resulted in reduced atherosclerosis in the pLDLR-LDLR-miR82 vector treated mice. Here we demonstrate for the first time, that an episomal non-viral vector is able to significantly reduce LDL-C and the progression of atherosclerosis in a mouse model of FH. Based on the success of the miRNA to further enhance the lipid lowering of our non-viral vector, we went on to screen small molecules which can drive the expression of the human LDLR promoter. 216 molecules were screened, where we identified a novel series of small molecules that increased the LDLR in human and mouse cell lines at nano molar potencies. We then went on to elucidate the mechanism of action as squalene synthase inhibitors and demonstrated, that when used in combination with statins, these compounds give a much greater increase in LDLR expression than statins alone. These small molecules could be used in conjunction with low dose statins, alongside our non-viral vector to further enhance expression of the LDLR and provide greater therapeutic outcome for patients of FH.

A novel peroxisome proliferator response element modulates hepatic low-density lipoprotein receptor gene transcription in response to PPARδ activation.

The hepatic expression of low-density lipoprotein (LDL) receptor (LDLR) gene is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein 2 (SREBP2). However whether additional cis-regulatory elements contribute to LDLR transcription has not been fully explored. We investigated the function of a putative peroxisome proliferator-activated receptor (PPAR)-response element (PPRE) sequence motif located at -768 to -752 bases upstream of the transcription start site of human LDLR gene in response to PPARδ activation. Promoter luciferase reporter analyses showed that treating HepG2 cells with PPARδ agonist L165041 markedly increased the activity of a full-length LDLR promoter construct (pLDLR-1192) without any effects on the shorter promoter reporter pLDLR-234 that contains only the core regulatory elements SRE-1 and SP1 sites. Importantly, mutation of the PPRE sequence greatly attenuated the induction of the full-length LDLR promoter activity by L165041 without affecting rosuvastatin (RSV)-mediated transactivation. EMSA and ChIP assay further confirmed the binding of PPARδ to the LDLR-PPRE site. Treating HepG2 cells with L165041 elevated the mRNA and protein expressions of LDLR without affecting the LDLR mRNA decay rate. The induction of LDLR expression by PPARδ agonist was further observed in liver tissue of mice and hamsters treated with L165041. Altogether, our studies identify a novel PPRE-mediated regulatory mechanism for LDLR transcription and suggest that combined treatment of statin with PPARδ agonists may have advantageous effects on LDLR expression.

Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins.

Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD–15 cells, which lack insulin-induced gene–1 (Insig–1) and Insig–2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity.

Alginate oligosaccharide enhances LDL uptake via regulation of LDLR and PCSK9 expression

The hepatic low-density lipoprotein (LDL) receptor (LDLR) plays a crucial role in lipoprotein metabolism by lowering the plasma LDL-cholesterol concentration, which reduces the risk for cardiovascular diseases. Although alginate oligosaccharide (AOS), prepared from degradation, has several pharmacological effects, it is not known whether AOS affects lipoprotein metabolism. This study was conducted to investigate whether AOS up-regulated LDLR expression and LDL uptake in vitro and in vivo, and the underlying molecular mechanism. We found that AOS increased LDLR expression and intracellular uptake of LDL by hepatocytes in a dose- and time-dependent manner. It is well established that sterol-responsive element binding protein-2 (SREBP-2) is an essential transcription factor for LDLR gene expression. AOS enhanced SREBP-2 nuclear translocation and mRNA levels. The specific role of SREBP-2 activation in AOS-induced LDLR expression was verified using an LDLR promoter construct with a sterol response element deletion. The activation of SREBP-2 by AOS is mediated by phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3β pathways. Furthermore, we found that expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), a crucial modulator of LDLR, was down-regulated by AOS; this related to the inhibition of hepatocyte nuclear factor-1α. Treatment of mice with AOS for 2 weeks stimulated LDLR expression and reduced PCSK9 expression, resulting in decreased plasma LDL-cholesterol levels. We conclude that AOS lowered plasma LDL-cholesterol levels through regulation LDLR expression. This effect was dependent on SREBP-2 and PCSK9.

Abstract 544: A Novel Peroxisome Proliferator Response Element Modulates LDL-receptor Transcription in Response to PPAR Agonists.

Hepatic LDL receptor (LDLR) binds to and internalizes circulating LDL-cholesterol thereby facilitating its clearance from the serum. LDLR is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein (SREBP1/2). Additionally, SRE-dependent modulation of LDLR by peroxisome proliferator-activated receptor gamma (PPARγ) has also been previously described. However whether additional promoter elements directly regulate LDLR transcription is not very well understood. We observed that treatment with the PPARδ agonist L165041 elevated luciferase-reported activity of human LDLR promoter as well as the mRNA and protein expression of LDLR in both, human and mouse cell lines. The induction of LDLR mRNA in HepG2 cells was dose-dependent with maximal expression observed at 20uM. We also observed that the half-life of LDLR mRNA was similar in control and L165041 treated samples, suggesting that the induction was a result of transactivation of mRNA synthesis. Combinatorial treatment of HepG2 cells with L165041 and rosuvastain (RSV) demonstrated additive effects of the two compounds on LDLR mRNA as well as protein expression. Transcription factor prediction programs indicated presence of a single PPAR-response element (PPRE)-like sequence motif in the human LDLR promoter, located at -768 to -752 bases upstream of the translation start site. Luciferase reporter analyses indicated that mutation or deletion of this PPRE-like element resulted in attenuation of L165041 mediated upregulation of LDLR promoter luciferase activity. Electrophoretic mobility shift assays further confirmed the binding of PPARδ/RXR heterodimers to the LDLR-PPRE site. Taken together, our studies identify a novel PPRE in the proximal promoter of LDLR and suggest that combinatorial treatment of statin with PPAR agonist may have advantageous effects on LDLR expression.

A single secreted luciferase-based gene reporter assay

Promoter analysis typically employs a reporter gene fused to a test promoter combined with a second reporter fused to a control promoter that is used for normalization purposes. However, this approach is not valid when experimental conditions affect the control promoter. We have developed and validated a single secreted luciferase reporter (SSLR) assay for promoter analysis that avoids the use of a control reporter. The approach uses an early level of expression of a secreted luciferase linked to a test promoter as an internal normalization control for subsequent analysis of the same promoter. Comparison of the SSLR assay with the dual luciferase reporter (DLR) assay using HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) and LDLR (low-density lipoprotein receptor) promoter constructs, which are down-regulated by 25-hydroxycholesterol, show that both assays yield similar results. Comparison of the response of the HMGCR promoter in SSLR transient assays compared very favorably with the response of the same promoter in the stable cell line. Overall, the SSLR assay proved to be a valid alternative to the DLR assay for certain applications and had significant advantages in that measurement of only one luciferase is required and monitoring can be continuous because cell lysis is not necessary.

A DNA Microarray for the Detection of Point Mutations and Copy Number Variation Causing Familial Hypercholesterolemia in Europe

To facilitate genetic cascade screening for familial hypercholesterolemia (FH) in Europe, two versions (7 and 9) of a DNA microarray were developed to detect the most frequent point mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin 9 (PCSK9) genes. The design of these microarrays is based on LIPOchip, version 4, which detects 191 LDLR and APOB mutations identified in Spanish patients with FH. A major improvement of LIPOchip, versions 7 and 9, is the ability to detect copy number variation (deletions or duplications of entire exons) in LDLR, thus abolishing the need to perform multiplex ligase-dependent probe amplification in patients with FH. The aim of this study was to validate a tool capable of detecting point mutations and copy number variations simultaneously and to evaluate its use and the newly developed software for analysis in clinical practice by reanalysis of several patients with known mutations causing FH. With the help of these validations, several aspects were analyzed, improved, and implemented in a newer version, which was evaluated through an internal validation.

Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T3

The technique of in vivo electroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp −612 and −156 were required for the l-triiodothyronine (T3) response. ChIP analysis showed that binding of TRβ1 to the −612 and −156 TREs was markedly stimulated by T3in vivo. Introduction of siRNAs against TRβ1/RXRα with LDL receptor promoter-luciferase construct by in vivo electroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cells in vitro thus validating this technique for in vivo studies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPARα and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3 treatment.

Transcriptional Activation of Low-Density Lipoprotein Receptor Gene by DJ-1 and Effect of DJ-1 on Cholesterol Homeostasis

DJ-1 is a novel oncogene and also causative gene for familial Parkinson’s disease park7. DJ-1 has multiple functions that include transcriptional regulation, anti-oxidative reaction and chaperone and mitochondrial regulation. For transcriptional regulation, DJ-1 acts as a coactivator that binds to various transcription factors, resulting in stimulation or repression of the expression of their target genes. In this study, we found the low-density lipoprotein receptor (LDLR) gene is a transcriptional target gene for DJ-1. Reduced expression of LDLR mRNA and protein was observed in DJ-1-knockdown cells and DJ-1-knockout mice and this occurred at the transcription level. Reporter gene assays using various deletion and point mutations of the LDLR promoter showed that DJ-1 stimulated promoter activity by binding to the sterol regulatory element (SRE) with sterol regulatory element binding protein (SREBP) and that stimulating activity of DJ-1 toward LDLR promoter activity was enhanced by oxidation of DJ-1. Chromatin immunoprecipitation, gel-mobility shift and co-immunoprecipitation assays showed that DJ-1 made a complex with SREBP on the SRE. Furthermore, it was found that serum LDL cholesterol level was increased in DJ-1-knockout male, but not female, mice and that the increased serum LDL cholesterol level in DJ-1-knockout male mice was cancelled by administration with estrogen, suggesting that estrogen compensates the increased level of serum LDL cholesterol in DJ-1-knockout female mice. This is the first report that DJ-1 participates in metabolism of fatty acid synthesis through transcriptional regulation of the LDLR gene.

Modified methylenedioxyphenol analogs lower LDL cholesterol through induction of LDL receptor expression

Although statin therapy is a cornerstone of current low density lipoprotein (LDL)-lowering strategies, there is a need for additional therapies to incrementally lower plasma LDL cholesterol. In this study, we investigated the effect of several methylenedioxyphenol derivatives in regulating LDL cholesterol through induction of LDL receptor (LDLR). INV-403, a modified methylenedioxyphenol derivative, increased LDLR mRNA and protein expression in HepG2 cells in a dose- and time-dependent fashion. These effects were apparent even under conditions of HMG-CoA reductase inhibition. Electrophoresis migration shift assays demonstrated that INV-403 activates SREBP2 but not SREBP1c, with immunoblot analysis showing an increased expression of the mature form of SREBP2. Knockdown of SREBP2 reduced the effect of INV-403 on LDLR expression. The activation of SREBP2 by INV-403 is partly mediated by Akt/GSK3β pathways through inhibition of phosphorylation-dependent degradation by ubiquitin-proteosome pathway. Treatment of C57Bl/6j mice with INV-403 for two weeks increased hepatic SREBP2 levels (mature form) and upregulated LDLR with concomitant lowering of plasma LDL levels. Transient expression of a LDLR promoter-reporter construct, a SRE-mutant LDLR promoter construct, and a SRE-only construct in HepG2 cells revealed an effect predominantly through a SRE-dependent mechanism. INV-403 lowered plasma LDL cholesterol levels through LDLR upregulation. These results indicate a role for small molecule approaches other than statins for lowering LDL cholesterol.