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A large number of metabolic waste products accumulate in the blood of patients with renal failure. Since these solutes have deleterious effects on the biological functions, they are called uremic toxins and have been classified in three groups: 1) small water soluble solutes (MW < 500 Da), 2) small solutes with known protein binding (MW < 500 Da), and 3) middle molecules (500 Da < MW < 60 kDa). Protein bound uremic toxins are poorly removed by conventional hemodialysis treatments because of their high protein binding and high distribution volume. The prototypical protein bound uremic toxins indoxyl sulfate (IS) and p-cresyl sulfate (pCS) are associated with the progression of chronic kidney disease, cardiovascular outcomes, and mortality of patients on maintenance hemodialysis. Furthermore, these two compounds are bound to albumin, the main plasma protein, via electrostatic and/or Van-der-Waals forces. The aim of the present thesis was to develop a dialysis strategy, based on the reversible modification of the ionic strength in the blood stream by increasing the sodium chloride (NaCl) concentration, in order to enhance the removal of protein bound substances, such as IS and pCS, with the ultimate goal to improve clinical patient outcomes. Enhancing the NaCl concentration ([NaCl]) in both human normal and uremic plasma was efficient to reduce the protein bound fraction of both IS and pCS by reducing their binding affinity to albumin. Increasing the ionic strength was feasible during modified pre-dilution hemodiafiltration (HDF) by increasing the [NaCl] in the substitution fluid. The NaCl excess was adequately removed within the hemodialyzer. This method was effective to increase the removal rate of both protein bound uremic toxins. Its ex vivo hemocompatibility, however, was limited by the osmotic shock induced by the high [NaCl] in the substituate. Therefore, modified pre-dilution HDF was further iterated by introducing a second serial cartridge, named the serial dialyzers (SDial) setup. This setting was validated for feasibility, hemocompatibility, and toxin removal efficiency. A better hemocompatibility at similar efficacy was obtained with the SDial setup compared with the modified pre-dilution HDF. Both methods were finally tested in an animal sheep model of dialysis to verify biocompatibility. Low hemolysis and no activation of both the complement and the coagulation systems were observed when increasing the [NaCl] in blood up to 0.45 and 0.60 M with the modified pre-dilution HDF and the SDial setup, respectively. In conclusion, the two dialysis methods developed to transitory enhance the ionic strength in blood demonstrated adequate biocompatibility and improved the removal of protein bound uremic toxins by decreasing their protein bound fraction. The concepts require follow-on clinical trials to assess their in vivo efficacy and their impact on long-term clinical outcomes.
DHEA is a precursor for the male and female sex hormones testosterone and estradiol, which are mainly secreted from the testes and the ovary, respectively. In addition, epidemiological studies showed that low serum levels of DHEA and DHEAS correlate with the incidence of autoimmune disease, cancer and cardiovascular disease. In vitro, DHEA and DHEAS influenced glucose metabolism in a favourable manner. However, positive effects of DHEA substitution were only significant adrenal insufficiency in women.
Steroid sulphotransferase 2A1 (SULT2A1) is the responsible enzyme for sulphonation of DHEA to DHEAS which is thought to be the inactive form of DHEA. In this role, SULT2A1 acts as a central regulator of steroid synthesis because sulphonation of DHEA withdraws the substrate for further downstream conversion. Another essential cofactor for sulphonation is PAPS, which is produced by the enzyme PAPS synthase (PAPSS) from ATP and anorganic sulphate. PAPSS exists in the different isoforms PAPSS1 and PAPSS2 and splice variants PAPSS2a and PAPSS2b. Changes in PAPSS activity are thought to influence sulphonation of DHEA significantly. However, neither regulation of PAPSS nor its influence on SULT2A1 have been investigated in human cell lines or humans.
The main goal of this thesis was to analyze the enzyme expression of the DHEA/DHEA shuttle, i.e. mRNA and protein of SULT2A1, PAPSS1 and PAPSS2, in various human cell lines. Furthermore, I investigated which cell line could serve as a suitable model for further research regarding regulation of SULT2A1, PAPSS1 and PAPSS2.
Here, I could show that the enzymes of the DHEA/DHEAS shuttle were expressed in the human adrenal cell line NCI-h295R as both mRNA and protein. In enzyme assays, I was able to prove conversion of DHEA to DHEAS as well as to different other steroids. However, applying Trilostane, a potent inhibitor of CYP3B, effectively directed conversion of DHEA to DHEAS. Using these findings, future experiments can investigate for example the influence of certain cytokines or endocrine disruptors on expression and activity of PAPSS1/2 and on sulphonation of DHEA. In particular, the relatively equal expression of PAPSS1 and PAPSS2 will enable us to do knock down experiments with siRNA to elucidate how the activity of one enzyme changes when the other one fails.
Sulphonation of DHEA by SULT2A1 is thought to happen in the cytoplasm or more precisely in the Golgi apparatus. However, experiments in transfected cells have shown both a cytoplasmatic and a nuclear localisation when both enzymes were expressed at the same time. Immunocytochemistry revealed the same results in the adrenal cell line NCI-h295R, where both enzymes were expressed strongly in the nucleus. The physiological role is not clear and requires further research. Presumably, sulphate is activated in the nucleus. However, one could also speculate that a shift of PAPSS to the nucleus could generate a reservoir, which can be activated by re-localisation to the cytoplasm when more PAPS is needed.
Expression of SULT2A1 in some foetal tissues has been investigated earlier. Whilst in adult human cartilage PAPSS1 is predominant, in newly born hamsters PAPSS2 is more abundantly expressed. The expression of PAPSS isoforms in highly sulphonating tissue has not been investigated in humans, so far. This work demonstrated a differential expression of SULT2A1, PAPSS1 and PAPSS2 in adult and foetal liver, adrenal and foetal cartilage tissue. In adult and foetal adrenal expression was similar. However, foetal and adult liver differed in the expression of SULT2A1, which was expressed much more in adult tissue. Most importantly, in foetal cartilage there was only a low expression of SULT2A1 and PAPS seems to mostly provided by PAPSS1, which was considerably higher expressed in cartilage than in other tissues. In contrast, PAPSS2 was mainly expressed in adult and foetal adrenal.
Additionally, we reported a case of a female patient who had been investigated for hyperandrogenism. Two mutations in the PAPSS2 gene had led to massively reduced serum levels of DHEAS. One heterozygous mutation in the domain of the APS kinase of the PAPSS2 protein leads to substitution of one amino acid at position 48 (T48R). In vitro experiments showed a residual activity of 6% for this mutation. A second mutation in the ATP sulphurylase domain of PAPSS2 was found. The introduction of thymidine instead of cytidine leads to a stop codon, which is presumed to truncate the protein at position 329 (R329X). In vitro, no residual activity was seen for this mutation. The lack of PAPS reduces sulphonation of DHEA but also sulphonation of proteoglycanes, which leads to skeletal abnormalities. The abundance of DHEA enables massive downstream conversion to androgens leading to clinical features of hyperandrogenism. Regarding the bone abnormalities, it is interesting and surprising that activity of PAPSS1 compensated to a great extent in cartilage but was not able to keep up a more considerable sulphonation of DHEA. Possibly, the subcellular localisation might play a role in this scenario.
Atherosclerosis is an active and progressive condition where the vascular cell adhesion molecules as VCAM-1 play a vital role controlling the recruitment of immune cells within the early and advanced plaques. Therefore targeting of VCAM-1 molecules with specific contrast agent bears the possibility to monitor the VCAM-1 expression, visualize the plaque progression starting at the early alterations, and help to establish early prevention of atherosclerosis before the origin of the thrombus formation, of which late recognition leads to myocardial infarction. Furthermore noninvasive magnetic resonance imaging (MRI) offers the benefit of combining the molecular and anatomic data and would thus enable specific detection of VCAM-1 targeted iron oxide contrast agent within inflammatory process of atherosclerosis. This thesis exactly presents the VCAM-1 concept as a suitable molecular approach and the potential of specific ultrasmall superparamagnetic iron oxide (USPIO) conjugated to the VCAM-1 binding peptide over unspecific non-targeted USPIO particles for evaluation of atherosclerosis. This work firstly demonstrated that selection of VCAM-1 molecules offers a good and potential strategy for imaging of atherosclerosis, as these vascular cell adhesion molecules are highly expressed in the early phase of inflammation and also continuously up-regulated within the advanced plaques. Secondly, this thesis showed the proof of principle and capability of the newly designed USPIO contrast agent conjugated to the specific cyclic peptide for VCAM-1 recognition. The experimental studies including ultra-high field MRI enabled further ex vivo and in vivo detection of applied USPIO-VCAM-1 particles within the aortic root region of early and advanced atherosclerotic plaques of 12 and 30 week old apolipoprotein E deficient (ApoE-/-) mice. Using a combination of histology and electron microscopy, this study for the first time pointed to distribution of targeted USPIO-VCAM-1 particles within plaque cells expressing VCAM-1 not only in luminal regions but also in deeper medial smooth muscle cell areas. Hence functionalized USPIO particles targeting VCAM-1 molecules allow specific and sensitive detection of early and advanced plaques at the molecular level, giving the new possibilities for early recognition of atherosclerotic plaques before the appearance of advanced and prone to rupture lesions. In contrast to the functionalized USPIO-VCAM-1, utilized non-targeted USPIO particles did not succeed in early plaque 6 identification limiting visualization of atherosclerosis to advanced forms in atherosclerotic ApoE-/- mice.
Isolated left ventricular non-compaction cardiomyopathy (LVNC) is a congenital myocardial disease characterized by excessive and prominent trabeculations in the left ventricle with deep intertrabecular recesses. Trabeculation is, however, a non specific finding which is present not only in LVNC but also in other cardiomyopathies like dilated cardiomyopathy (DCM) and even in healthy controls, therefore, differential diagnosis keeps puzzling clinicians. Therefore the present study aimed to comprehensively explore regional myocardial deformation properties in adult patients with isolated LVNC using strain and strain rate imaging derived from tissue Doppler imaging and 2D speckle tracking. It was proposed that the knowledge of deformation properties in LVNC would help to differentiate patients with LVNC and DCM. A total of 14 patients with LVNC, 15 patients with DCM, and 15 healthy controls were included in this study. The groups were matched for age and gender. Standard 2D echocardiography was performed in all subjects, and tissue Doppler imaging (TDI) of all ventricular walls was acquired using parasternal long axis, apical 4-chamber, 2-chamber, and apical long axis views. Deformation imaging data derived from both TDI and grey scale images were analyzed. Clinical and standard echocardiographic findings in patients with LVNC and DCM were similar. In patients with LVNC, hypertrabeculation was mostly located in the apical and mid segments of the left ventricle and strikingly more than in patients with DCM. The extent of non-compaction was poorly related to global left ventricular systolic function (LVEF) as well as regional myocardial function assessed by strain rate imaging. Regional myocardial systolic deformation in patients with LVNC was significantly impaired in the left and right ventricles in both longitudinal and radial direction. There was a striking difference on longitudinal myocardial systolic function between LVNC and DCM patients, i.e., an increasing strain and strain rate gradient from apex to base in patients with LVNC, whereas patients with DCM displayed a homogeneously decreased strain and strain rate in all segments. Results derived from 2D speckle tracking method were consistent with those from TDI method. Analysis of myocardial mechanical asynchrony revealed a lack of myocardial contraction synchrony in the LVNC and DCM patients. The time to systolic peak velocity was obviously delayed in these two patient groups. However, the mechanical asynchrony features were similar in patients with LVNC and DCM and could not serve for differential diagnosis. In conclusion, LVNC and DCM are both cardiomyopathies presenting reduced regional myocardial function and mechanical asynchrony. Nevertheless differential diagnosis can be made by analysis of hypertrabeculation as well as analysis of regional myocardial deformation pattern.
Cardiovascular disease is the most common mortality risk in the industrialized world. Myocardial infarction (MI) results in the irreversible loss of cardiac muscle, triggering pathophysiological remodelling of the ventricle and development of heart failure. Insufficient myocardial capillary density within the surviving myocardium after MI has been identified as a critical event in this process, although the underlying molecular signalling pathways of cardiac angiogenesis are mechanistically not well understood. The discovery of microRNAs (miRNAs, miRs), small non-coding RNAs with 19-25 nucleotides in length, has introduced a new level of the regulation of cardiac signalling pathways. MiRNAs regulate gene expression post-transcriptionally by binding to their complementary target messenger RNAs (mRNAs) and represent promising therapeutic targets for gene therapy. Here, it is shown that cardiac miR-24 is primarily expressed in cardiac endothelial cells and upregulated following MI in mice and hypoxic conditions in vitro. Enhanced miR-24 expression induces endothelial cell apoptosis and impairs endothelial capillary network formation. These effects on endothelial cell biology are at least in part mediated through targeting of transcription factor GATA2, histone deacetylase H2A.X, p21-activated kinase PAK4 and Ras p21 protein activator RASA1. Mechanistically, target repression abolishes respective and secondary downstream signalling cascades. Here it is shown that endothelial GATA2 is an important mediator of cell cycle, apoptosis and angiogenesis at least in part by regulation of cytoprotective heme oxygenase 1 (HMOX1). Moreover, additional control of endothelial apoptosis is achieved by the direct miR-24 target PAK4. Its kinase function is essential for anti-apoptotic Bad phosphorylation in endothelial cells. In a mouse model of MI, blocking of endothelial miR-24 by systemic administration of a specific antagonist (antagomir) enhances capillary density in the infarcted heart and preserves cardiac function. The current findings indicate miR-24 to act as a critical regulator of endothelial cell apoptosis and angiogenesis. Modulation of miR-24 may be potentially a suitable strategy for therapeutic intervention in the setting of ischemic heart diseases.
Summary: I previously demonstrated that conditional overexpression of the neuronal nitric oxide synthase (nNOS) inhibited L-type Ca2+-channels and decreased myocardial contractility1 (Burkard N. et al. (2007). Circ Res 100, 32-44). However, nNOS has multiple targets within the cardiac myocyte and it is possible that interesting biological functions of this protein remain to be elucidated. In this study, I showed that nNOS overexpression has a cardioprotective effect after ischemia-reperfusion injury by inhibiting mitochondrial function and reducing the generation of reactive oxygen species (ROS). The effect of conditional nNOS overexpression in cardiac myocytes in ischemiareperfusion injury was assessed. Ischemia-reperfusion injury in WT mice resulted in nNOS accumulation in the mitochondria. Similary, transgenic nNOS overexpression caused nNOS abundance in mitochondria. Electron microscopy of mouse myocardium from nNOS overexpressing mice showed that after induction of its expression, nNOS is additionally localised in mitochondria. nNOS translocation into mitochondria was dependent on HSP90. Ischemia-reperfusion experiments in isolated hearts showed a cardioprotective effect of nNOS overexpression (30min post-ischemia, LVDP 27.0±2.5mmHg in non-induced animals vs. 45.2±1.9mmHg in nNOS overexpressing mice, n=12, p<0.05). Consistently with this finding, in vivo the infarct size within the area at risk was significantly decreased in nNOS overexpressing mice compared to non-induced animals (36.6±8.4 relative % vs. 61.1±2.9 relative %, n=12, p<0.05). nNOS overexpression also caused a significant increase in mitochondrial nitrite levels accompanied by a decrease of cytochrome c oxidase activity (72.0±8.9units/ml in nNOS overexpressing mice vs. 113.2±17.1units/ml in non-induced mice, n=12, p<0.01) resulting in an inhibition of mitochondrial function. Accordingly, O2-consumption (MVO2) in isolated heart muscle stripes was decreased in nNOS overexpressing mice, already under resting conditions (0.016±0.0015 vs. 0.024±0.006ml[O2] x mm-3 x min-1, n=13, p<0.05). Additionally, this study showed that the ROS concentration was significantlydecreased in hearts of nNOS overexpressing mice compared to non-induced animals (6.14±0.685 vs. 14.53±1.7μM, n=8, p<0.01). Application of different inhibitors, Western Blot analysis and activity assays showed that the lower ROS concentration in nNOS overexpressing mice was caused by inhibition of the xanthine oxidoreductase (XOR) activity by the increased abundance of nNOS expression. In summary, this study demonstrated that the conditional transgenic overexpression of nNOS resulted in myocardial protection after ischemia-reperfusion injury. Besides reduction of myocardial Ca2+-overload after reperfusion this might be caused by inhibition of mitochondrial function through nNOS, which reduced myocardial oxygen consumption already under baseline conditions (Burkard N. conditionally accepted by
Free fatty acids (FFA) modulate the effectiveness of glucose to suppress endogenous glucose production (EGP), and increased FFA levels contribute importantly to the loss of glucose effectiveness in type 2 diabetes mellitus (T2DM). Elevating FFA levels in nondiabetic (ND) subjects for at least 6h both increases gluconeogenesis (GNG) and impairs glucose effectiveness. Therefore, we wished to define the extent to which an increase in GNG is responsible for the loss of glucose effectiveness and whether EGP can be inhibited in the presence of elevated plasma FFA by inhibiting GNG with ethanol. To determine the effect of inhibiting GNG on glucose effectiveness, EGP ([3-3H]-glucose) was measured during three separate 7h normoglycemic/hyperglycemic pancreatic clamp studies (somatostatin; basal glucagon/GH/insulin replacement) in n=7 ND subjects (1F/6M; age=45±5 yr; BMI=27.6±3.0 kg/m2). Following an initial 210 min interval of euglycemia (5 mmol/l), blood glucose levels were raised to hyperglycemic levels (10 mmol/l) from t=210-420 min. The first pancreatic clamp study was a baseline study with saline infusions (Lip-/Et-). Lipid emulsion (Liposyn 20%) was infused throughout the second and third study types (Lip+ and Lip+/Et+) to increase FFA to T2DM levels (~ 500 mmol/l). In addition to Liposyn, ethanol (Et) was infused during hyperglycemia in the third study type (Lip+/Et+), using a pharmacokinetic algorithm to attain GNG-inhibiting ethanol levels of 80 mg/dl within 20 min. Under baseline conditions, hyperglycemia suppressed EGP by 61%. After raising plasma FFA to T2DM levels, suppression of EGP by hyperglycemia was impaired in Lip+ (34% decrease). During the Lip+/Et+ co-infusion studies the infusion of ethanol enhanced suppression of EGP by hyperglycemia (65.8% decrease, P=0.004 vs. Lip+) and thus restored glucose effectiveness (P=0.6 vs. Lip-/Et-). Thus, our results confirm the striking effects of elevated plasma FFA to impair glucose effectiveness and suggest that increased GNG contributes importantly to this loss of regulation. Inhibiting GNG could be an effective means of lowering EGP and improving glucose effectiveness in T2DM.
The incidence of cardiovascular diseases including cardiac hypertrophy and failure in pre-menopausal women is lower compared to age-matched men but the risk of heart disease increases substantially after the onset of menopause. It has been postulated that female sex hormones play an important role in cardiovascular health in pre-menopausal women. In animal studies including spontaneously hypertensive (SHR) rats, the development of cardiac hypertrophy is attenuated by 17β-estradiol treatment. Cardiac energy metabolism is crucial for normal function of the heart. In cardiac hypertrophy and heart failure, the myocardium undergoes a metabolic shift from fatty acid as primary cardiac energy source to glucose, which re-introduces the fetal type of metabolism that representing the glucose as a major source of energy. Many studies have reported that the disruption of the balance between glucose and fatty acid metabolism plays an important role in cardiac pathologies including hypertrophy, heart failure, diabetes, dilative cardiomyopathy and myocardial infarction. Glucose enters cardiomyocytes via GLUT1 and GLUT4 glucose transporters and GLUT4 is the major glucose transporter which is insulin-dependent. Cardiac-selective GLUT4 deficiency leads to cardiac hypertrophy. This shows that the decrease in cardiac glucose uptake may play a direct role in the pathogenesis of cardiac hypertrophy. Estrogens modulate glucose homeostasis in the liver and the skeletal muscle. But it is not known whether estrogens affect also cardiac glucose uptake which could provide another mechanism to explain the prevention of cardiac hypertrophy by female sex hormones. In the present study, SHR Rats were ovariectomized (OVX), not ovariectomized (sham) or ovariectomized and treated with subcutaneous 17β-estradiol. After 6 weeks of treatment, body weight, the serum levels of estrogen, insulin, intra-peritoneal glucose tolerance test (IP-GTT), myocardial glucose uptake by FDG-PET (2-(18F)-fluoro-deoxyglucose (18FDG) and Positron Emission Tomography), cardiac glucose transporter expression and localization and cardiac hexokinase activity were analyzed. As results of this study, PET analysis of female SHR revealed decreased cardiac glucose uptake in OVX animals compared to intact that was normalized by estrogen supplementation. Interestingly, there was no change in global glucose tolerance among the treatment groups. Serum insulin levels and cardiac hexokinase activity were elevated by E2 substitution. The protein content of cardiac glucose transporters GLUT-4 and GLUT-1, and their translocation as determined by fractionation studies and immuno-staining did not show any significant change by ovariectomy and estrogen replacement. Also levels of insulin receptor substrate-1 (IRS-1) and its tyrosine phosphorylation, which is required for activation and translocation of GLUT4, was un-affected in all groups of SHR. Cardiac gene expression analysis in SHR heart showed that ei4Ebp1 and Frap1 genes which are involved in the mTOR signaling pathway, were differentially expressed upon estrogen treatment. These genes are known to be activated in presence of glucose in the heart. As a conclusion of this study, reduced myocardial FDG uptake in ovariectomized spontaneously hypertensive rat is normalized by 17β-estradiol treatment. Increased myocardial hexokinase appears as a potential mechanism to explain increased myocardial glucose uptake by 17β-estradiol. Increased cardiac glucose uptake in response to 17β-estradiol in ovariectomized SHR may provide a novel mechanism to explain the reduction of cardiac hypertrophy in E2 treated SHR. Therefore, 17β-estradiol improves cardiac glucose utilization in ovariectomized SHR which may give rise to possible mechanism for its protective effects against cardiac hypertrophy.
Insight into oxidative stress mediated by nitric oxide synthase (NOS) isoforms in atherosclerosis
(2008)
The principle product of each NOS is nitric oxide. However, under conditions of substrate and cofactor deficiency the enzymes directly catalyze superoxide formation. Considering this alternative chemistry of each NOS, the effects of each single enzyme on key events of atherosclerosis are difficult to predict. Here, we evaluate nitric oxide and superoxide production by all three NOS isoforms in atherosclerosis. ESR measurements of circulating and vascular wall nitric oxide production showed significantly reduced nitric oxide levels in apoE/eNOS double knockout (dko) and apoE/iNOS dko animals but not in apoE/nNOS dko animals suggesting that eNOS and iNOS majorly contribute to vascular nitric oxide production in atherosclerosis. Pharmacological inhibition and genetic deletion of eNOS and iNOS reduced vascular superoxide production suggesting that eNOS and iNOS are uncoupled in atherosclerotic vessels. Though genetic deletion of nNOS did not alter superoxide production, acute inhibition of nNOS showed that nNOS contributes significantly to superoxide production. In conclusion, uncoupling of eNOS occurs in apoE ko atherosclerosis but eNOS mediated superoxide production does not outweigh the protective effects of eNOS mediated nitric oxide production. We show that although nNOS is not a major contributor of the vascular nitric oxide formation, it prevents atherosclerosis development. Acute inhibition of nNOS showed a significant reduction of superoxide formation suggesting that nNOS is uncoupled. The exact mechanism of action of nNOS in atheroprotection is yet to be elucidated. Genetic deletion of iNOS reduced NADPH oxidase activity. Thus, iNOS has both direct and indirect proatherosclerotic effects, as it directly generates both nitric oxide and superoxide simultaneously resulting in peroxynitrite formation and indirectly modulates NADPH oxidase activity. We hypothesize that eNOS is coupled in the disease free regions of the vessel and contributes to nitric oxide generation whereas in the diseased region of the vessel it is uncoupled to produce superoxide (Figure 16). nNOS expressed in the smooth muscle cells of the plaque contributes to the local superoxide generation. iNOS expressed in smooth muscle cells and leukocytes of the plaque generates superoxide and nitric oxide simultaneously to produce the strong oxidant peroxynitrite.
Inhibition of Nuclear Import of Calcineurin Prevents the Development of Myocardial Hypertrophy
(2007)
The Calcineurin/NFAT signaling cascade is a crucial transducer of cellular function. It has recently been emerged that in addition to the transcription factor NFAT, the phosphatase Calcineurin is also translocated to the nucleus. Our traditional understanding of Calcineurin activation via sustained high Ca2+-levels was also advanced by recent findings from this working group (AG Ritter), which showed that Calcineurin is activated by proteolysis of the C-terminal autoinhibitory domain. This leads to the constitutive activation and nuclear translocation of Calcineurin. Therefore, Calcineurin is not only responsible for dephosphorylating of NFAT in the cytosol thus enabling its nuclear import, its presence in the nucleus is also significant in ensuring the full transcriptional activity of NFAT. Formation of complexes between transcription factors and DNA regulates the transcriptional process. Therefore, the time that transcription factors remain nuclear is a major determinant of transcriptional activity. The movement of proteins over ~40 kDa into and out of the nucleus is governed by the nuclear pore complex (NPC). Transcription factors and enzymes that regulate the activity of these proteins are shuttled across the nuclear envelope by proteins that recognize nuclear localization signals (NLS) and nuclear export signals (NES) within the amino acid sequence of these transcription factors. In this study, the precise mechanisms of Calcineurin nuclear import and export were identified. Additionally to the nuclear localization sequence (NLS) and the nuclear export sequence (NES) within the sequence of Calcineurin, the respective nuclear cargo proteins, responsible for nuclear import, Importinβ1, and for nuclear export, CRM1, were identified. Inhibition of the Calcineurin/importin interaction by a competitive peptide, called Import Blocking Peptide (IBP), which mimicked the Calcineurin NLS, prevented nuclear entry of Calcineurin. A non-inhibitory control peptide showed no effect. Using this approach, it was able to prevent the development of myocardial hypertrophy. In Angiotensin II stimulated cardiomyocytes, both the transcriptional and the translational level was suppressed. Additionally, cell size and expression of Brain natriuretic peptide (as molecular marker for hypertrophy) were significantly reduced compared untreated controls. IBP worked dose-dependent, but did not affect the Calcineurin phosphatase activity. In conclusion, Calcineurin is not only capable of dephosphorylating NFAT, thus enabling its nuclear import, its presence in the nucleus is also important for full NFAT transcriptional activity. Using IBP to prevent the nuclear import of Calcineurin is a completely new approach to prevent the development of myocardial hypertrophy.
The first goal of this study was to develop cell lines with a stable expression of bio-fluorescent topo II and topo I. This was successfully achieved using a bicistronic vector system. Control experiments showed that proteins of expected size were expressed, and that GFP-tagged topos I, IIa, and IIb were active in the cells and fully integrated in the endogenous pools of the enzymes. These cell-lines provided a novel tool for investigating the cell biology of human DNA topoisomerases. Our most important finding was, that both types of mammalian topoisomerases are entirely mobile proteins that are in continuous and rapid flux between all compartments of the nucleus and between the cytososl and the chromosomes of mitotic cells. This was particularly surprising with regard to topo II, which is considered to be a structural component of the nuclear matrix and the chromosome scaffold. We must conclude that if this was the case, then these architectural structures appear to be much more dynamic than believed until now. In this context it should also be mentioned, that the alignment of topo II with the central axes of the chromosome arms, which has until now been considered a hall-mark of the enzyme’s association with the chromosomal scaffold, is not seen in vivo and can be demonstrated to be to some extent an artefact of immunohistochemistry. Furthermore, we show that the two isoforms of topo II (a and b) have a different localisation during mitotic cell division, supporting the general concept that topo II functions at mitosis are exclusively assigned to the a-form, whereas at interphase the two isoenzymes work in concert. Despite unrestricted mobility within the entire nuclear space, topoisomerases I and II impose as mostly nucleolar proteins. We show that this is due to the fact that in the nucleoli they are moving slower than in the nucleoplasm. The decreased nucleolar mobility cannot be due to DNA-interactions, because compounds that fix topoisomerases to the DNA deplete them from the nucleoli. Interestingly, the subnucleolar distribution of topoisomerases I and II was complementary. The type II enzyme filled the entire nucleolar space, but excluded the fibrial centers, whereas topo I accumulated at the fibrial centers, an allocation directed by the enzyme’s N-terminus. During mitosis, it also mediates association with the nucleolar organising regions of the acrocentric chromosomes. Thus, topo I stays associated with the rDNA during the entire cell-cycle and consistently colocalizes there with RNA-polymerase I. Finally, we show that certain cancer drugs believed to act by stabilising covalent catalytic DNA-intermediates of topoisomerases, do indeed immobilize the enzymes in living cells. Interestingly, these drugs do not target topoisomerases in the nucleoli but only in the nucleoplasm.
Two isoforms of human CD23 (CD23a and CD23b) have been described. They differ by only 6-7 residues in the N-terminal cytoplasmic tail. CD23a is restrictively expressed on B-cells while CD23b is inducible on B-cells, as well as monocytes, eosinophils, macrophages and a variety of other cell types, after IL-4 stimulation. The two isoforms seems to have different functions. CD23a appears to be the isoform associated with endocytosis of IgE immune complexes and mediating antigen presentation on B-cells. CD23b has a phagocytosis motif and seems to be involved in the phagocytosis of IgE-coated particles, cytokine release and the generation of superoxides. Previous studies indicate that the two isoforms connect to different signal transduction pathways. Comparing the cells that express only one or both CD23 isoforms suggests that CD23b is involved in upregulating cAMP and iNOS, whereas CD23a mediates an increase in intracellular calcium. In the main part of the study we investigated how the CD23a B-cell specific expression is regulated. Pax-5 is a B-cell restricted transcription factor with an essential role in early and late B-cell development. Putative Pax-5 binding sites have been predicted in the CD23a proximal promoter. Analyses of the CD23a promoter revealed three putative Pax-5 binding sites with more than 50% homology to the consensus sequence. One of these sites, named CD23-1 can compete a high affinity Pax-5 binding site or can directly bind Pax-5 protein in electrophoretic mobility shift assays. Introducing mutations into this site abrogates the binding. A different approach, in which overlapping peptides covering the length of the CD23a promoter were tested in competition assays against a high affinity binding site, also revealed CD23-1 as the only site that directly binds Pax-5 protein. Expression of Pax-5 in 293 cells resulted in a 7-fold activation of a CD23a core promoter construct. Co-transfection together with STAT6 showed that Pax-5 cooperates with this transcription factor in enhancing the level of transcription of a CD23a extended promoter construct. Most importantly, ectopic expression of Pax-5 in the monocytic cell line U-937 that regularly expresses only the CD23b isoform enabled a significant CD23a expression after stimulation with IL-4 and PMA. Our results suggest that Pax-5 is a key regulator of the B-cell restricted expression of the CD23a isoform. In the second part of the project, we used a yeast two-hybrid system (CytoTrapTM from Stratagene) in order to look for cytoplasmic interaction partners for the CD23 receptor. The system was established in order to reach a high efficiency of transformation and different bait vector constructs were made. The screening was performed using a human spleen library cloned in the target vector of the system. The first bait constructs used (pSosCD23a and pSosCD23b) expressed the very short (22 amino acids) cytoplasmic tails of the isoforms at the C-terminal end of the fusion protein (human SOS). Improved bait constructs, (pSosCD23a+Linker and pSos CD23b+Linker) expressed the cytoplasmic tail of CD23a/b at the N-terminal side of the human SOS and had in consequence the N-terminal part free as a bait, as it occurs in vivo. A flexible linker region separated the fusion proteins in order to make the small amino acid bait chain more obvious. Approximately three million library clones were screened with these various constructs. No “true positive” interaction was detected. A relatively high number of “false positive” clones were obtained and checked in another two-hybrid system. A new bait construct, in which the tyrosine residue in the cytoplasmic tail of CD23a was replaced by a glutamic acid residue will be used for future screening. The system was also used in order to test the interaction between CD23 and p59fyn, a member of the Src family of protein kinases that was mentioned to associate with CD23a. No interaction was detected by using the CytoTrap two-hybrid system. In conclusion, the key result of the study demonstrates that Pax-5 is a main regulator of the B-cell specific expression of the CD23a isoform. In addition, a two-hybrid system was established and employed in order to look for cytoplasmic interaction partners for CD23.
Unique functions of DNA topoisomerase IIalpha and IIbeta have been suggested. A human cell line which carries a homozygeous mutation of the nuclear localization sequence of the topoisomerase IIalpha gene expresses the isoform outside the nucleus at the onset of mitosis. At mitosis topoisomerase IIbeta diffused away from the chromatin despite the nuclear lack of the IIalpha-form. Chromosome condensation and disjunction was performed with the aid of cytosolic topoisomerase IIalpha which bound to the mitotic chromatin with low affinity. Consequently an increased rate of nondisjunction is observed in these cells. It is concluded that high affinity chromatin binding of topoisomerase IIalpha is essential for chromosome condensation/disjunction and that topoisomerase IIbeta does not adopt these functions. A centrosomal protein was recognized by topoisomerase IIalpha. This topoisomerase IIalpha-like protein resembles a modified form of topoisomerase IIalpha with an apparent size of 205 kDa compared to 170 kDa. The expression of the protein is constant in all stages of the cell cycle and it appears in proliferating as well as in resting cells. If there is not sufficient topoisomerase IIalpha present at mitosis the centrosomal proteins might adopt the function and a mitotic catastrophe in the cells could therefore be prevented.