Hedi Gierens

Interleukin-6 Stimulates LDL Receptor Gene Expression via Activation of Sterol-Responsive and Sp1 Binding Elements

Inflammatory or malignant diseases are associated with elevated levels of cytokines and abnormal low density lipoprotein (LDL) cholesterol metabolism. In the acute-phase response to myocardial injury or other trauma or surgery, total and LDL cholesterol levels are markedly decreased. We investigated the effects of the proinflammatory cytokine interleukin (IL)-6 on LDL receptor (LDL-R) function and gene expression in HepG2 cells. IL-6 dose-dependently increased the binding, internalization, and degradation of (125)I-LDL. IL-6-stimulated HepG2 cells revealed increased steady-state levels of LDL-R mRNA. In HepG2 cells transiently transfected with reporter gene constructs harboring the sequence of the LDL-R promoter extending from nucleotide -1563 (or from nucleotide -234) through -58 relative to the translation start site, IL-6 dose-dependently increased promoter activity. In the presence of LDL, a similar relative stimulatory effect of IL-6 was observed. Studies using a reporter plasmid with a functionally disrupted sterol-responsive element (SRE)-1 revealed a reduced stimulatory response to IL-6. In gel-shift assays, nuclear extracts of IL-6-treated HepG2 cells showed an induced binding of SRE binding protein (SREBP)-1a and SRE binding protein(SREBP)-2 to the SRE-1 that was independent of the cellular sterol content and an induced binding of Sp1 and Sp3 to repeat 3 of the LDL-R promoter. Our data indicate that IL-6 induces stimulation of the LDL-R gene, resulting in enhanced gene transcription and LDL-R activity. This effect is sterol independent and involves, on the molecular level, activation of nuclear factors binding to SRE-1 and the Sp1 binding site in repeat 2 and repeat 3 of the LDL-R promoter, respectively.

Effect of atorvastatin, simvastatin, and lovastatin on the metabolism of cholesterol and triacylglycerides in HepG2 cells.

We evaluated the effects of the hydroxymethylglutaryl coenzyme A reductase inhibitors (HMGRI) atorvastatin, lovastatin, and simvastatin on lipid homeostasis in HepG2 cells. The drugs were almost equally effective in inhibiting cholesterol synthesis and in decreasing cellular cholesterol. Atorvastatin and lovastatin increased low-density lipoprotein receptor mRNA (2.5-fold at 3 x 10(-7) M) and the transcription rate at the promoter of the low-density lipoprotein receptor gene (>5-fold at 10(-6) M). The three compounds enhanced the activity of the low-density lipoprotein receptor at a similar magnitude (1.6-2.1- fold at 10(-6) M). Atorvastatin and lovastatin increased the nuclear form of sterol regulatory element binding protein (SREBP)-2, but not of SREBP-1. Each of the drugs increased triacylglyceride synthesis (50% at 10(-7)-10(-6) M), cellular triacylglyceride content (16% at 10(-6) M), and expression of fatty acid synthase by reporter gene and Northern blot analysis (2-fold and 2.7-fold at 10(-6) M and 3 x 10(-7) M, respectively). All compounds reduced the secretion of apo B (30% at 3 x 10(-7) M). HMGRI decreased the ratio of cholesterol to apo B in newly synthesised apo B containing particles by approximately 50% and increased the ratio of triacylglycerides to apo B by approximately 35%. We conclude that regulatory responses to HMGRI are mediated by SREBP-2 rather than by SREBP-1, that HMGRI oppositely affect the cellular cholesterol and triacylglyceride production, that HMGRI moderately decrease the release of apo B containing particles, but profoundly alter their composition, and that atorvastatin does not significantly differ from other HMGRI in these regards.

Rapid genotyping of human platelet antigen 5 with fluorophore-labelled hybridization probes on the LightCycler.

Genotyping of human platelet alloantigens (HPA) has become an important procedure in the diagnosis and prevention of disorders such as neonatal alloimmune thrombocytopenic purpura, post‐transfusion purpura, and refractoriness to platelet transfusion therapy. We present a single‐tube method for HPA‐1 genotyping that combines rapid‐cycle PCR with allele‐specific fluorescent probe melting profiles for product genotyping. A fragment covering the polymorphic site is amplified in the presence of two fluorescently‐labelled hybridization probes. During the annealing step of the thermal cycling, both probes bind to their complementary sequences in the amplicon resulting in resonance energy transfer, thus providing real‐time fluorescence monitoring of PCR. Continuous aquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in an allele‐specific manner as the detection probe, which was fully complementary to the HPA‐1b allele, melted off the template. By determining the temperature at which maximum melting of the hybrids occurred, the two alleles were readily distinguishable. Using this method, genotyping of 32 samples was completed within 30 min without the need for any post‐PCR sample manipulation, thereby eliminating the risks of end‐product contamination and sample tracking errors. The genotypes determined with the LightCyclerTM were identical when compared with a conventional PCR and restriction fragment length polymorphism technique. The genotyping of HPA‐1 on the LightCycler is a rapid and reliable method that is suitable for typing both small and large numbers of samples.

Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labeled hybridization probes on the lightcycler™

Genotyping of human platelet alloantigens (HPA) has become an important procedure in the diagnosis and prevention of disorders such as neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura, and refractoriness to platelet transfusion therapy. We present a single-tube method for HPA-1 genotyping that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping. A fragment covering the polymorphic site is amplified in the presence of two fluorescently-labelled hybridization probes. During the annealing step of the thermal cycling, both probes bind to their complementary sequences in the amplicon resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous aquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in an allele-specific manner as the detection probe, which was fully complementary to the HPA-1b allele, melted off the template. By determining the temperature at which maximum melting of the hybrids occurred, the two alleles were readily distinguishable. Using this method, genotyping of 32 samples was completed within 30 min without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The genotypes determined with the LightCyclerTM were identical when compared with a conventional PCR and restriction fragment length polymorphism technique. The genotyping of HPA-1 on the LightCycler is a rapid and reliable method that is suitable for typing both small and large numbers of samples.

Rapid, Homogeneous Genotyping of Human Platelet Antigen 1 by Fluorescence Resonance Energy Transfer and Probe Melting Curves

Alloantigenic determinants on platelet membrane glycoproteins can be targets for alloimmune antibody responses that cause bleeding disorders such as neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura (PTP), and refractoriness to platelet transfusion therapy [1]. In PTP, a transfused patient forms alloantigen-specific antibodies that, for still unknown reasons, may cause destruction of autologous platelets often resulting in life-threatening thrombocytopenia. In feto-maternal alloimmune thrombocytopenia, designated as neonatal alloimmune thrombocytopenic purpura (NATP), fetal thrombocytopenia is caused by maternal alloimmunization against one or more paternal platelet alloantigens with similar life-threatening complications. Severe NATP may lead to either prenatal or neonatal intracranial haemorrhage that may cause fetal death or psychomoter impairment.