Heiko Funke-Kaiser

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.

Direct Angiotensin II Type 2 Receptor Stimulation Acts Anti-Inflammatory Through Epoxyeicosatrienoic Acid and Inhibition of Nuclear Factor κB

Angiotensin II type 2 (AT2) receptors can be regarded as an endogenous repair system, because the AT2 receptor is upregulated in tissue damage and mediates tissue protection. A potential therapeutic use of this system has only recently come within reach through synthesis of the first selective, orally active, nonpeptide AT2 receptor agonist, compound 21 (C21; dissociation constant for AT2 receptor: 0.4 nM; dissociation constant for angiotensin II type 1 receptor: >10 000 nM). This study tested AT2 receptor stimulation with C21 as a potential future therapeutic approach for the inhibition of proinflammatory cytokines and of nuclear factor &kgr;B. C21 dose-dependently (1 nM to 1 &mgr;mol/L) reduced tumor necrosis factor-&agr;–induced interleukin 6 levels in primary human and murine dermal fibroblasts. AT2 receptor specificity was controlled for by inhibition with the AT2 receptor antagonist PD123319 and by the absence of effects in AT2 receptor–deficient cells. AT2 receptor–coupled signaling leading to reduced interleukin 6 levels involved inhibition of nuclear factor &kgr;B, activation of protein phosphatases, and synthesis of epoxyeicosatrienoic acid. Inhibition of interleukin 6 promoter activity by C21 was comparable in strength to inhibition by hydrocortisone. C21 also reduced monocyte chemoattractant protein 1 and tumor necrosis factor-&agr; in vitro and in bleomycin-induced toxic cutaneous inflammation in vivo. This study is the first to show the anti-inflammatory effects of direct AT2 receptor stimulation in vitro and in vivo by the orally active, nonpeptide AT2 receptor agonist C21. These data suggest that pharmacological AT2 receptor stimulation may be an orally applicable future therapeutic approach in pathological settings requiring the reduction of interleukin 6 or inhibition of nuclear factor &kgr;B.

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.

The past, present and future of angiotensin II type 2 receptor stimulation

Studying the angiotensin type 2 receptor (AT2) has been problematic in the past because a pharmacological tool for direct, specific in vitro and in vivo stimulation of the receptor has been lacking. Consequently, current knowledge about AT2 receptor signalling and function had to be obtained by indirect approaches, like studying animals or cells with genetically altered AT2 receptor expression levels, inhibitory experiments using specific AT2 receptor antagonists, stimulation with angiotensin II under concomitant angiotensin II type 1 receptor blockade or stimulation with the peptide agonist CGP42112A, which has additional AT2 receptor antagonistic properties. The recently developed non-peptide AT2 receptor agonist Compound 21 now, for the first time, allows direct, selective and specific AT2 receptor stimulation in vitro and in vivo . This new tool will certainly revolutionise AT2 receptor research, enable many new insights into AT2 receptor function and may also have the potential to become a future medical drug. This article reviews milestone findings about AT2 receptor functional properties obtained by ‘conventional’ experimental approaches within the last 20 years. Moreover, it provides an overview of the first results obtained by direct AT2 receptor stimulation with Compound 21, comprising effects on alkaline secretion, neurite outgrowth, blood pressure and post-infarct cardiac function.

Adapter proteins and promoter regulation of the angiotensin AT2 receptor — implications for cardiac pathophysiology

The angiotensin AT 2 receptor (AT2R) represents an important component of the renin-angiotensin system since it is involved in the (patho) physiology of different cardiovascular and neuronal diseases. Furthermore, AT2 receptors can partly mediate beneficial effects of angiotensin AT 1 receptor (AT1R) blockers, and direct pharmacological AT 2 receptor agonism emerges as a novel therapeutic strategy. This review discusses the constitutive and ligand-mediated activity as well as the signal transduction of the AT2 receptor, focusing on adapter proteins which directly bind to this receptor. Direct protein-protein interaction partners of the AT2 receptor described so far include the transcription factor promyelocytic zinc finger protein, AT2 receptor binding protein and the AT1 receptor. In addition, the putative crosstalk of the AT2 receptor with the renin/ prorenin receptor (RER) via the promyelocytic zinc finger protein (PLZF) and the role of oestrogens on the regulation of the AT2 receptor are presented. Conceiving the coupling of the AT2 receptor to different adapter proteins with distinct and partly opposing cellular effects and the implications of its constitutive activity might help to overcome the current controversies on the (patho)physiological role of the AT2 receptor.

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.

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.