Jan H. Schefe

Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s C T difference” formula

For quantification of gene-specific mRNA, quantitative real-time RT-PCR has become one of the most frequently used methods over the last few years. This article focuses on the issue of real-time PCR data analysis and its mathematical background, offering a general concept for efficient, fast and precise data analysis superior to the commonly used comparative CT (ΔΔCT) and the standard curve method, as it considers individual amplification efficiencies for every PCR. This concept is based on a novel formula for the calculation of relative gene expression ratios, termed GED (Gene Expression’s CT Difference) formula. Prerequisites for this formula, such as real-time PCR kinetics, the concept of PCR efficiency and its determination, are discussed. Additionally, this article offers some technical considerations and information on statistical analysis of real-time PCR data.

A Novel Signal Transduction Cascade Involving Direct Physical Interaction of the Renin/Prorenin Receptor With the Transcription Factor Promyelocytic Zinc Finger Protein

A human renin/prorenin receptor (RER) has recently been cloned. To gain insight into the molecular function of the RER, we studied its signal transduction mechanisms. Initially, we found a ubiquitous and intracellular expression pattern of the human RER. Consistently, we observed several transcriptional start sites and a high promoter activity of the human RER. We could identify the transcription factor promyelocytic zinc finger (PLZF) protein as a direct protein interaction partner of the C-terminal domain of the RER by yeast 2-hybrid screening and coimmunoprecipitation. Coimmunoprecipitation experiments also indicated homodimerization of the RER. On activation of the RER by renin, PLZF is translocated into the nucleus and represses transcription of the RER itself, thereby creating a very short negative feedback loop, but activates transcription of the p85α subunit of the phosphatidylinositol-3 kinase (PI3K-p85α). Small interfering RNA against the RER abolished these effects. A PLZF cis-element in the RER promoter was identified by site-directed mutagenesis and electrophoretic mobility-shift assay. Renin stimulation caused a 6-fold recruitment of PLZF to this promoter region as shown by chromatin immunoprecipitation. Moreover, renin stimulation of rat H9c2 cardiomyoblasts induced an increase of cell number and a decrease of apoptosis. These effects were partly abolished by PI3K inhibition and completely abrogated by small interfering RNA against PLZF. Finally, experiments in PLZF knockout mice confirmed the role of PLZF as an upstream regulator of RER and PI3K-p85α. Our data demonstrate the existence of a novel signal transduction pathway involving the ligand renin, RER, and the transcription factor PLZF, which is of physiological and putative pathophysiological relevance.

Regulation of Transport of the Angiotensin AT2 Receptor by a Novel Membrane-Associated Golgi Protein

Objective— Synthesis and maturation of G protein–coupled receptors are complex events that require an intricate combination of processes including protein folding, posttranslational modifications, and transport through distinct cellular compartments. Little is known concerning the regulation of G protein–coupled receptor transport from the endoplasmic reticulum to the cell surface. Methods and Results— Here we show that the cytoplasmatic carboxy-terminal of the angiotensin AT2 receptor (AT2R) acts independently as an endoplasmic reticulum–export signal. Using a yeast two-hybrid system, we identified a Golgi membrane–associated protein termed ATBP50 (for AT2R binding protein of 50 kDa) that binds to this motif. We also cloned ATBP60 and ATBP135 encoded by the same gene as ATBP50 that mapped to chromosomes 8p21.3. Downregulation of ATBP50 using siRNA leads to retention of AT2R in inner compartments, reduced cell surface expression, and decreased antiproliferative effects of the receptor. Conclusion— Our results indicate that ATBP50 regulates the transport of the AT2R to cell membrane by binding to a specific motif within its cytoplasmic carboxy-terminal and thereby enabling the antiproliferative effects of the receptor.

Prorenin engages the (pro)renin receptor like renin and both ligand activities are unopposed by aliskiren

Objectives Inhibition of (pro)renin receptor activation was demonstrated to inhibit or even abolish the development of end-organ damage in animal models. The new renin inhibitor, aliskiren, markedly increases the plasma concentration of the (pro)renin receptor ligands prorenin and renin in patients. The effects of prorenin and of renin inhibitors on the signal transduction cascade of the (pro)renin receptor are currently unknown. Results Our results indicate that renin and prorenin were equally potent in (pro)renin receptor activation by decreasing (pro)renin receptor mRNA, increasing phosphatidylinositol-3 kinase p85α mRNA and augmenting viable cell number, respectively. These effects of renin and prorenin are both abolished using small-interfering RNA against the (pro)renin receptor or its adaptor promyelocytic zinc finger protein. The renin inhibitor aliskiren did not inhibit the renin-induced or prorenin-induced activation of the (pro)renin receptor. Conclusion This is the first report demonstrating equal ligand activities of both, renin and prorenin, on the (pro)renin receptor - promyelocytic zinc finger protein–phosphatidylinositol-3 kinase–p85α pathway. The failure of aliskiren to inhibit the noncatalytic effects of renin and prorenin may be of clinical relevance considering the increase in plasma concentrations of (pro)renin under aliskiren treatment.

The beta-lactam antibiotic, ceftriaxone, dramatically improves survival, increases glutamate uptake and induces neurotrophins in stroke.

Objective Ceftriaxone has been reported to reduce neuronal damage in amyotrophic lateral sclerosis and in an in-vitro model of neuronal ischaemia through increased expression and activity of the glutamate transporter, GLT1. We tested the effects of ceftriaxone on mortality, neurological outcome, and infarct size in experimental stroke in rats and looked for underlying mechanisms. Methods Male normotensive Wistar rats received ceftriaxone (200 mg/kg intraperitoneal) as a single injection 90 min after middle cerebral artery occlusion (90 min with reperfusion). Forty-eight hours after middle cerebral artery occlusion, infarct size (MRI) and neurological deficits were estimated. GLT1 expression was determined by real time RT-PCR, immunoblotting and promoter reporter assay, astrocyte GLT1 activity by measuring glutamate uptake. Bacterial load in various organs was measured by real time RT-PCR, neurotrophins and IL-6 by immunoblotting. Results Ceftriaxone dramatically reduced early (24-h) mortality from 34.5% (vehicle treatment, n = 29) to 0% (P < 0.01, n = 19). In a subgroup, followed up for 4 weeks, mortality persisted at 0%. Ceftriaxone strongly tended to reduce infarct size, it significantly improved neuronal survival within the penumbra, reduced neurological deficits (P < 0.001) and led to an upregulation of neurotrophins (P < 0.01) in the peri-infarct zone. Ceftriaxone did not increase GLT1 expression, but increased GLT1 activity (P < 0.05). Conclusion Ceftriaxone causes a significant reduction in acute stroke mortality in a poststroke treatment regimen in animal studies. Improved neurological performance and survival may be due to neuroprotection by activation of GLT1 and a stimulation of neurotrophins resulting in an increased number of surviving neurons in the penumbra.

Signal transduction of the (pro)renin receptor as a novel therapeutic target for preventing end-organ damage

The (pro)renin receptor ((P)RR) not only represents a novel component of the renin–angiotensin system but is also a promising novel drug target because of its crucial involvement in the pathogenesis of renal and cardiac end-organ damage. This review discusses the signal transduction of the (P)RR with its adapter protein promyelocytic zinc-finger protein, the impact of this receptor, especially on cardiovascular disease, and its putative interaction with renin inhibitors such as aliskiren. Furthermore, the increasing complexity regarding the cellular function of the (P)RR is addressed, which arises by the intimate link with proton pumps and the phosphatase PRL-1, as well as by the presence of different subcellular localizations and of a soluble isoform of the (P)RR. Finally, the rationale and strategy for the development of small-molecule antagonists of the (P)RR, called renin/prorenin receptor blockers, are presented.

Comparison between single and combined treatment with candesartan and pioglitazone following transient focal ischemia in rat brain.

Angiotensin AT1 receptor blockers (ARBs) and thiazolidinediones (TZDs) have become well established drugs for the treatment of major risk factors of stroke. Since several studies provided evidence that ARBs and TZDs also have additional anti-inflammatory effects, we hypothesized that a combined treatment with the ARB, candesartan, and the TZD, pioglitazone, ameliorates ischemia-induced brain injury and inflammation by synergistic anti-inflammatory actions. Normotensive Wistar rats were pre-treated for 5 days with vehicle (0.9% NaCl), 0.2 mg/kg/day candesartan (s.c.), and/or 2 and/or 20 mg/kg/day pioglitazone (p.o.), respectively and underwent 90 min of middle cerebral artery occlusion (MCAO) with successive reperfusion. Neurological deficits and infarct size were determined 24 h and 48 h after MCAO, respectively, followed by tissue sampling. Animals treated with candesartan, pioglitazone, and the combination of candesartan and pioglitazone had reduced neurological deficits 24 h and 48 h after MCAO, respectively (P<0.05-0.01). Infarct size was reduced by treatment of candesartan, pioglitazone, and their respective combination (each P<0.05) 48 h after stroke compared to vehicle. Treatment with candesartan, pioglitazone, and their combination resulted in significantly reduced mRNA expression of the inflammatory markers CXCL1 and TNFalpha in vivo (P<0.01). The combination of candesartan plus pioglitazone is equally effective compared to their single applications concerning neuroprotection and attenuation of inflammation after MCAO. Therefore, we conclude that a direct synergistic neuroprotective action of parallel ARB and TZD treatment is unlikely.

PLZF and the (pro)renin receptor

For many years, angiotensin II with its respective receptors was considered to be the only effector molecule within the renin–angiotensin system. Nevertheless, several studies indicated that renin (the enzyme catalyzing the generation of angiotensin I) and its enzymatically inactive precursor prorenin may have an angiotensin-II-independent (patho)physiological significance. In 2002, a specific (pro)renin receptor ((P)RR)) which increases the enzymatic activity of its ligands and induces an intrinsic activity upon ligand binding has been published. Recently, our group has demonstrated a novel (P)RR signal transduction pathway involving direct protein–protein interaction between the (P)RR and the transcription factor promyelocytic zinc finger protein (PLZF) and the nuclear translocation of PLZF upon renin stimulation. Downstream effects of (P)RR activation by renin included repression of the (P)RR itself and induction of the p85α subunit of the phosphatidylinositol-3 kinase (PI3K-p85α) as well as an increase in proliferation and a decrease in apoptotic activity. Various animal models demonstrated that inhibition of prorenin binding to the (P)RR can prevent or even abolish the development of cardiac fibrosis and diabetic nephropathy via angiotensin-II-independent mechanisms. Additional studies that verify these remarkable findings are needed. Moreover, the potency of aliskiren (the first orally active renin inhibitor in the market) to interfere with a putatively detrimental binding of (pro)renin to the (P)RR is of particular interest and has to be elucidated.

The Promyelocytic Leukemia Zinc Finger (PLZF) Protein Exerts Neuroprotective Effects in Neuronal Cells and is Dysregulated in Experimental Stroke

Stroke is one of the major medical burdens in industrialized countries. Animal experiments indicate that blockade of the angiotensin AT1 receptor (AT1R) improves neurological outcome after cerebral ischemia. These protective effects are partially mediated by the angiotensin AT2 receptor (AT2R). The transcription factor promyelocytic leukemia zinc finger (PLZF) was identified as a direct adapter protein of the AT2R. Furthermore, our group was able to demonstrate that PLZF also directly binds and mediates the effects of the human (pro)renin receptor [(P)RR] which is involved in brain development. Therefore, we hypothesized that PLZF is involved in neuroprotection.

A Polymorphic Microsatellite Repeat within the ECE-1c Promoter Is Involved in Transcriptional Start Site Determination, Human Evolution, and Alzheimer's Disease

Genetic factors strongly contribute to the pathogenesis of sporadic Alzheimers disease (AD). Nevertheless, genome-wide association studies only yielded single nucleotide polymorphism loci of moderate importance. In contrast, microsatellite repeats are functionally less characterized structures within our genomes. Previous work has shown that endothelin-converting enzyme-1 (ECE-1) is able to reduce amyloid β content. Here we demonstrate that a CpG-CA repeat within the human ECE-1c promoter is highly polymorphic, harbors transcriptional start sites, is able to recruit the transcription factors poly(ADP-ribose) polymerase-1 and splicing factor proline and glutamine-rich, and is functional regarding haplotype-specific promoter activity. Furthermore, genotyping of 403 AD patients and 444 controls for CpG-CA repeat length indicated shifted allelic frequency distributions. Sequencing of 245 haplotype clones demonstrated that the overall CpG-CA repeat composition of AD patients and controls is distinct. Finally, we show that human and chimpanzee [CpG]m–[CA]n ECE-1c promoter repeats are genetically and functionally distinct. Our data indicate that a short genomic repeat structure constitutes a novel core promoter element, coincides with human evolution, and contributes to the pathogenesis of AD.