Publications by authors named "Lisann Pelzl"

39 Publications

Targeting Lyn Kinase in Chorea-Acanthocytosis: A Translational Treatment Approach in a Rare Disease.

J Pers Med 2021 May 10;11(5). Epub 2021 May 10.

Department of Neurology, University Hospital Carl Gustav Carus, Technische Universität Dresden, 01307 Dresden, Germany.

Chorea-acanthocytosis (ChAc) is a neurodegenerative disease caused by mutations in the gene. It is characterized by several neurological symptoms and the appearance of acanthocytes. Elevated tyrosine kinase Lyn activity has been recently identified as one of the key pathophysiological mechanisms in this disease, and therefore represents a promising drug target. We evaluated an individual off-label treatment with the tyrosine kinase inhibitor dasatinib (100 mg/d, 25.8-50.4 weeks) of three ChAc patients. Alongside thorough safety monitoring, we assessed motor and non-motor scales (e.g., MDS-UPDRS, UHDRS, quality of life) as well as routine and experimental laboratory parameters (e.g., serum neurofilament, Lyn kinase activity, actin cytoskeleton in red blood cells). Dasatinib appeared to be reasonably safe. The clinical parameters remained stable without significant improvement or deterioration. Regain of deep tendon reflexes was observed in one patient. Creatine kinase, serum neurofilament levels, and acanthocyte count did not reveal consistent effects. However, a reduction of initially elevated Lyn kinase activity and accumulated autophagy markers, as well as a partial restoration of the actin cytoskeleton, was found in red blood cells. We report on the first treatment approach with disease-modifying intention in ChAc. The experimental parameters indicate target engagement in red blood cells, while clinical effects on the central nervous system could not be proven within a rather short treatment time. Limited knowledge on the natural history of ChAc and the lack of appropriate biomarkers remain major barriers for "clinical trial readiness". We suggest a panel of outcome parameters for future clinical trials in ChAc.
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http://dx.doi.org/10.3390/jpm11050392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150322PMC
May 2021

Antibody-mediated procoagulant platelets in SARS-CoV-2-vaccination associated immune thrombotic thrombocytopenia.

Haematologica 2021 08 1;106(8):2170-2179. Epub 2021 Aug 1.

Anaesthesiology and Intensive Care Medicine, University Hospital Tuebingen.

The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. To prevent severe infection, mass COVID-19 vaccination campaigns with several vaccine types are currently underway. We report pathological and immunological findings in 8 patients who developed vaccine-induced immune thrombotic thrombocytopenia (VITT) after administration of SARS-CoV-2 vaccine ChAdOx1 nCoV-19. We analyzed patient material using enzyme immune assays, flow cytometry and heparin-induced platelet aggregation assay and performed autopsies on two fatal cases. Eight patients (5 female, 3 male) with a median age of 41.5 years (range, 24 to 53) were referred to us with suspected thrombotic complications 6 to 20 days after ChAdOx1 nCoV-19 vaccination. All patients had thrombocytopenia at admission. Patients had a median platelet count of 46.5 x109/L (range, 8 to 92). Three had a fatal outcome and 5 were successfully treated. Autopsies showed arterial and venous thromboses in various organs and the occlusion of glomerular capillaries by hyaline thrombi. Sera from VITT patients contain high titer antibodies against platelet factor 4 (PF4) (OD 2.59±0.64). PF4 antibodies in VITT patients induced significant increase in procoagulant markers (P-selectin and phosphatidylserine externalization) compared to healthy volunteers and healthy vaccinated volunteers. The generation of procoagulant platelets was PF4 and heparin dependent. We demonstrate the contribution of antibody-mediated platelet activation in the pathogenesis of VITT.
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http://dx.doi.org/10.3324/haematol.2021.279000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8327736PMC
August 2021

Therapeutic targeting of Lyn kinase to treat chorea-acanthocytosis.

Acta Neuropathol Commun 2021 05 3;9(1):81. Epub 2021 May 3.

Department of Neuroscience, Biomedicine and Movement Sciences, University of Verona, Verona, Italy.

Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, γ-synuclein and phospho-tau proteins in Vps13a basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a Lyn showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients.
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http://dx.doi.org/10.1186/s40478-021-01181-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091687PMC
May 2021

Antibody-induced procoagulant platelets in severe COVID-19 infection.

Blood 2021 02;137(8):1061-1071

Department of Anesthesiology and Intensive Care Medicine, University Hospital of Tuebingen, Tuebingen, Germany.

The pathophysiology of COVID-19-associated thrombosis seems to be multifactorial. We hypothesized that COVID-19 is accompanied by procoagulant platelets with subsequent alteration of the coagulation system. We investigated depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytosolic calcium (Ca2+) concentration, and phosphatidylserine (PS) externalization. Platelets from COVID-19 patients in the intensive care unit (ICU; n = 21) showed higher ΔΨm depolarization, cytosolic Ca2+, and PS externalization compared with healthy controls (n = 18) and non-ICU COVID-19 patients (n = 4). Moreover, significant higher cytosolic Ca2+ and PS were observed compared with a septic ICU control group (ICU control; n = 5). In the ICU control group, cytosolic Ca2+ and PS externalization were comparable with healthy controls, with an increase in ΔΨm depolarization. Sera from COVID-19 patients in the ICU induced a significant increase in apoptosis markers (ΔΨm depolarization, cytosolic Ca2+, and PS externalization) compared with healthy volunteers and septic ICU controls. Interestingly, immunoglobulin G fractions from COVID-19 patients induced an Fcγ receptor IIA-dependent platelet apoptosis (ΔΨm depolarization, cytosolic Ca2+, and PS externalization). Enhanced PS externalization in platelets from COVID-19 patients in the ICU was associated with increased sequential organ failure assessment score (r = 0.5635) and D-dimer (r = 0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared with those without. The strong correlations between markers for apoptosic and procoagulant platelets and D-dimer levels, as well as the incidence of thrombosis, may indicate that antibody-mediated procoagulant platelets potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients.
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http://dx.doi.org/10.1182/blood.2020008762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7791311PMC
February 2021

DJ-1 (Park7) affects the gut microbiome, metabolites and the development of innate lymphoid cells (ILCs).

Sci Rep 2020 09 30;10(1):16131. Epub 2020 Sep 30.

Institute of Medical Genetics and Applied Genomics, Tübingen University, Calwerstraße 7, 72076, Tübingen, Germany.

The proper communication between gut and brain is pivotal for the maintenance of health and, dysregulation of the gut-brain axis can lead to several clinical disorders. In Parkinson's disease (PD) 85% of all patients experienced constipation many years before showing any signs of motor phenotypes. For differential diagnosis and preventive treatment, there is an urgent need for the identification of biomarkers indicating early disease stages long before the disease phenotype manifests. DJ-1 is a chaperone protein involved in the protection against PD and genetic mutations in this protein have been shown to cause familial PD. However, how the deficiency of DJ-1 influences the risk of PD remains incompletely understood. In the present study, we provide evidence that DJ-1 is implicated in shaping the gut microbiome including; their metabolite production, inflammation and innate immune cells (ILCs) development. We revealed that deficiency of DJ-1 leads to a significant increase in two specific genera/species, namely Alistipes and Rikenella. In DJ-1 knock-out (DJ-1) mice the production of fecal calprotectin and MCP-1 inflammatory proteins were elevated. Fecal and serum metabolic profile showed that malonate which influences the immune system was significantly more abundant in DJ-1 mice. DJ-1 appeared also to be involved in ILCs development. Further, inflammatory genes related to PD were augmented in the midbrain of DJ-1 mice. Our data suggest that metabolites and inflammation produced in the gut could be used as biomarkers for PD detection. Perhaps, these metabolites and inflammatory mediators could be involved in triggering inflammation resulting in PD pathology.
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http://dx.doi.org/10.1038/s41598-020-72903-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528091PMC
September 2020

DJ-1 (Park7) affects the gut microbiome, metabolites and the development of innate lymphoid cells (ILCs).

Sci Rep 2020 09 30;10(1):16131. Epub 2020 Sep 30.

Institute of Medical Genetics and Applied Genomics, Tübingen University, Calwerstraße 7, 72076, Tübingen, Germany.

The proper communication between gut and brain is pivotal for the maintenance of health and, dysregulation of the gut-brain axis can lead to several clinical disorders. In Parkinson's disease (PD) 85% of all patients experienced constipation many years before showing any signs of motor phenotypes. For differential diagnosis and preventive treatment, there is an urgent need for the identification of biomarkers indicating early disease stages long before the disease phenotype manifests. DJ-1 is a chaperone protein involved in the protection against PD and genetic mutations in this protein have been shown to cause familial PD. However, how the deficiency of DJ-1 influences the risk of PD remains incompletely understood. In the present study, we provide evidence that DJ-1 is implicated in shaping the gut microbiome including; their metabolite production, inflammation and innate immune cells (ILCs) development. We revealed that deficiency of DJ-1 leads to a significant increase in two specific genera/species, namely Alistipes and Rikenella. In DJ-1 knock-out (DJ-1) mice the production of fecal calprotectin and MCP-1 inflammatory proteins were elevated. Fecal and serum metabolic profile showed that malonate which influences the immune system was significantly more abundant in DJ-1 mice. DJ-1 appeared also to be involved in ILCs development. Further, inflammatory genes related to PD were augmented in the midbrain of DJ-1 mice. Our data suggest that metabolites and inflammation produced in the gut could be used as biomarkers for PD detection. Perhaps, these metabolites and inflammatory mediators could be involved in triggering inflammation resulting in PD pathology.
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http://dx.doi.org/10.1038/s41598-020-72903-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528091PMC
September 2020

Beta-Glycerophosphate-Induced ORAI1 Expression and Store Operated Ca Entry in Megakaryocytes.

Sci Rep 2020 02 3;10(1):1728. Epub 2020 Feb 3.

Department of Vegetative and Clinical Physiology, Eberhard Karl University Tuebingen, Tuebingen, Germany.

Impairment of renal phosphate elimination in chronic kidney disease (CKD) leads to enhanced plasma and tissue phosphate concentration, which in turn up-regulates transcription factor NFAT5 and serum & glucocorticoid-inducible kinase SGK1. The kinase upregulates ORAI1, a Ca-channel accomplishing store-operated Ca-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by Ca-sensors STIM1 and/or STIM2. In megakaryocytes and blood platelets SOCE and thus ORAI1 are powerful regulators of activity. The present study explored whether the phosphate-donor ß-glycerophosphate augments NFAT5, ORAI1,2,3 and/or STIM1,2 expressions and thus SOCE in megakaryocytes. Human megakaryocytic Meg01cells were exposed to 2 mM of phosphate-donor ß-glycerophosphate for 24 hours. Platelets were isolated from blood samples of patients with impaired kidney function or control volunteers. Transcript levels were estimated utilizing q-RT-PCR, cytosolic Ca-concentration ([Ca]) by Fura-2-fluorescence, and SOCE from increase of [Ca] following re-addition of extracellular Ca after store depletion with thapsigargin (1 µM). NFAT5 and ORAI1 protein abundance was estimated with Western blots. As a result, ß-glycerophosphate increased NFAT5, ORAI1/2/3, STIM1/2 transcript levels, as well as SOCE. Transcript levels of NFAT5, SGK1, ORAI1/2/3, and STIM1/2 as well as NFAT5 and ORAI1 protein abundance were significantly higher in platelets isolated from patients with impaired kidney function than in platelets from control volunteers. In conclusion, phosphate-donor ß-glycerophosphate triggers a signaling cascade of NFAT5/SGK1/ORAI/STIM, thus up-regulating store-operated Ca-entry.
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http://dx.doi.org/10.1038/s41598-020-58384-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997179PMC
February 2020

Autoantibody-mediated desialylation impairs human thrombopoiesis and platelet lifespan.

Haematologica 2021 01 1;106(1):196-207. Epub 2021 Jan 1.

Transfusion Medicine, Medical Faculty of Tübingen, University Hospital Tübingen.

Immune thrombocytopenia is a common bleeding disease caused by autoantibody-mediated accelerated platelet clearance and impaired thrombopoiesis. Accumulating evidence suggests that desialylation affects platelet life span in immune thrombocytopenia. Herein, we report on novel effector functions of autoantibodies from immune thrombocytopenic patients which might interfere with the clinical picture of the disease. Data from our study show that a subgroup of autoantibodies is able to induce cleave of sialic acid residues from the surface of human platelets and megakaryocytes. Moreover, autoantibody-mediated desialylation interferes with the interaction between cells and extracellular matrix proteins leading to impaired platelet adhesion and megakaryocyte differentiation. Using a combination of ex vivo model of thrombopoiesis, a humanized animal model, and a clinical cohort study, we demonstrate that cleavage of sialic acid induces significant impairment in production, survival as well as function of human platelets. These data may indicate that prevention of desialylation should be investigated in the future in clinical studies as a potential therapeutic approach to treat bleeding in immune thrombocytopenia.
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http://dx.doi.org/10.3324/haematol.2019.236117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7776251PMC
January 2021

Blood donor-derived buffy coat to produce platelets in vitro.

Vox Sang 2020 Jan 10;115(1):94-102. Epub 2019 Nov 10.

Medical Faculty of Tübingen, University of Tübingen, Tübingen, Germany.

Background And Objectives: Platelet transfusion is a standard medical therapy used to treat several bleeding disorders. However, a critical drawback is the dependency on donor-derived platelets, which leads to concerns like insufficient availability and immunological complications. In vitro platelet production from hematopoietic progenitor cells (CD34) may represent a reasonable solution.

Materials And Methods: CD34+ cells were isolated from either buffy coat or peripheral blood and compared in terms of platelet production in vitro. The number and the quality of magnetically isolated CD34+ cells and their capability to differentiate into mature megakaryocytes were investigated using flow cytometry. Additionally, the functionality of megakaryocytes in term of in vitro platelet production was tested.

Results: Similar purity and quantity of CD34+ cells was found after their isolation from both cell sources. In contrast, after 6 days of culture, enhanced number of CD34+ cells isolated from buffy coat compared with peripheral blood was observed (5·3 x 106 vs. 3·0 x 106, respectively). Interestingly, despite a comparable nuclear maturation phenotype, the yield of platelets released from buffy coat-derived megakaryocytes was significantly higher than from peripheral blood cells (platelet yield pro MK: 7·2 vs. 2·7, respectively). Importantly, platelets produced from buffy coat-derived cells could be activated by agonists.

Conclusion: Haematopoietic progenitor cells isolated from buffy coat have increased yield of platelets released from mature megakaryocytes and enhanced in vitro functionality, compared with peripheral blood-derived cells. Our study, suggests that buffy coat, obtained during blood donation processing, might be a promising source of megakaryocytes for in vitro platelet production.
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http://dx.doi.org/10.1111/vox.12863DOI Listing
January 2020

Gut Bacterial Metabolite Urolithin A (UA) Mitigates Ca Entry in T Cells by Regulating miR-10a-5p.

Front Immunol 2019 31;10:1737. Epub 2019 Jul 31.

Department of Physiology, University of Tübingen, Tübingen, Germany.

The gut microbiota influences several biological functions including immune responses. Inflammatory bowel disease is favorably influenced by consumption of several dietary natural plant products such as pomegranate, walnuts, and berries containing polyphenolic compounds such as ellagitannins and ellagic acid. The gut microbiota metabolizes ellagic acid resulting in the formation of bioactive urolithins A, B, C, and D. Urolithin A (UA) is the most active and effective gut metabolite and acts as a potent anti-inflammatory and anti-oxidant agent. However, whether gut metabolite UA affects the function of immune cells remains incompletely understood. T cell proliferation is stimulated by store operated Ca entry (SOCE) resulting from stimulation of Orai1 by STIM1/STIM2. We show here that treatment of murine CD4 T cells with UA (10 μM, 3 days) significantly blunted SOCE in CD4 T cells, an effect paralleled by significant downregulation of Orai1 and STIM1/2 transcript levels and protein abundance. UA treatment further increased miR-10a-5p abundance in CD4 T cells in a dose dependent fashion. Overexpression of miR-10a-5p significantly decreased STIM1/2 and Orai1 mRNA and protein levels as well as SOCE in CD4 T cells. UA further decreased CD4 T cell proliferation. Thus, the gut bacterial metabolite UA increases miR-10a-5p levels thereby downregulating Orai1/STIM1/STIM2 expression, store operated Ca entry, and proliferation of murine CD4 T cells.
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http://dx.doi.org/10.3389/fimmu.2019.01737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685097PMC
September 2020

Evaluation of a flow cytometer-based functional assay using platelet-rich plasma in the diagnosis of heparin-induced thrombocytopenia.

Thromb Res 2019 Aug 31;180:55-61. Epub 2019 May 31.

Center for Clinical Transfusion Medicine Tuebingen, Germany; Medical Faculty of Tuebingen, University of Tuebingen, Germany. Electronic address:

Background: Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4/heparin (PF4/hep)-complexes. The in vitro demonstration of PF4/hep antibodies using functional assays is essential for an optimal management of patients suspected to have HIT. However, conventional functional assays are technically challenging and limited to specialized laboratories. In contrast, flow cytometers are commonly used in routine laboratories. The aim of this study is to investigate the performance characteristics of a commercially available, flow cytometer based assay in the diagnosis of HIT.

Study Design: Sera of consecutive patients with suspected HIT were investigated using the Emo-test HIT Confirm® assay and compared to the standard method consisting of an IgG-specific enzyme immunoassay (EIA) for anti-PF4/hep antibodies and the heparin induced platelet aggregation (HIPA) test.

Results: 390 sera were included in the study, 164 sera tested IgG EIA-positive, of which 33 also tested HIPA-positive. No HIPA-positive samples were EIA-negative. In the Emo-test HIT Confirm® assay, 112 sera revealed positive results (%Hepla > 13); however, 51 (45.5%) were EIA-negative. Of the 33 HIPA-positive/EIA-positive HIT sera, 23 tested positive in the Emo-test HIT Confirm® assay, 2 gave ambiguous results, and 8 sera yielded false-negative results. Accordingly, the HIT Confirm® assay showed a sensitivity of 69.7% with a slightly better specificity of 75.4% compared to the EIA (sensitivity 100%, specificity 63.3%). An increase in diagnostic specificity for HIT to 85% was found when positive results were obtained in both the Emo-test HIT Confirm® assay and EIA.

Conclusion: The Emo-Test HIT Confirm® assay may improve the specificity of laboratory investigations of HIT. However, the assay can only be recommended in combination with an immunoassay due to the high rate of false negativity. Our observation indicates a need to establish external quality assessment for functional assays to avoid such clinically relevant pitfalls.
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http://dx.doi.org/10.1016/j.thromres.2019.05.016DOI Listing
August 2019

The assembly and evaluation of antisense oligonucleotides applied in exon skipping for titin-based mutations in dilated cardiomyopathy.

J Mol Cell Cardiol 2019 06 15;131:12-19. Epub 2019 Apr 15.

Department of Cardiology and Cardiovascular Diseases, Eberhard Karls University, Tübingen, Germany; Department of Invasive Electrophysiology, RWTH Aachen, Pauwelsstr. 30, Aachen, Germany. Electronic address:

The leading cause of genetic dilated cardiomyopathy (DCM) is due to mutations in the TTN gene, impacting approximately 15-20% of familial and 18% of sporadic DCM cases. Currently, there is potential for a personalized RNA-based therapeutic approach in titin-based DCM, utilizing antisense oligonucleotide (AON) mediated exon-skipping, which attempts to reframe mutated titin transcripts, resulting in shortened, functional protein. However, the TTN gene is massive with 363 exons; each newly identified TTN exon mutation provides a challenge to address when considering the potential application of AON mediated exon skipping. In the initial phase of this strategy, the mutated TTN exon requires specific AON design and evaluation to assess the exon skipping effectiveness for subsequent experiments. Here, we present a detailed protocol to effectively assemble and evaluate AONs for efficient exon-skipping in targeted TTN exons. We chose a previously identified TTN 1-bp deletion mutation in exon 335 as an exemplary target exon, which causes a frameshift mutation leading to truncated A-band titin in DCM. We designed two specific AONs to mask the Ttn exon 335 and confirmed successfully mediated exon skipping without disrupting the Ttn reading frame. In addition, we evaluated and confirmed AON-treated HL-1 cells show maintained store-operated calcium entry, fractional shortening as well as preserved sarcomeric formation in comparison to control samples, indicating the treated cardiomyocytes retain adequate, essential cell function and structure, proving the treated cells can compensate for the loss of exon 335. These results indicate our method offers the first systematic protocol in designing and evaluating AONs specifically for mutated TTN target exons, expanding the framework of future advancements in the therapeutic potential of antisense-mediated exon skipping in titin-based DCM.
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http://dx.doi.org/10.1016/j.yjmcc.2019.04.014DOI Listing
June 2019

Upregulation of Orai1 and STIM1 expression as well as store-operated Ca entry in ovary carcinoma cells by placental growth factor.

Biochem Biophys Res Commun 2019 05 20;512(3):467-472. Epub 2019 Mar 20.

Department of Internal Medicine III, Eberhard Karls,University, Tübingen, Germany. Electronic address:

Placental growth factor (PlGF) is produced by tumor cells and stimulates tumor growth and metastasis in part by upregulation of hypoxia inducible factor HIF1α. Orchestration of tumor cell proliferation and migration involves oscillations of cytosolic Ca activity ([Ca]). The [Ca] oscillations could be accomplished by triggering of intracellular Ca release followed by store-operated Ca-entry (SOCE). Mechanisms accomplishing SOCE include the pore-forming ion channel unit Orai1 and its regulator STIM1. The present study explored whether PlGF influences the expression of Orai1 and STIM1, as well as SOCE and whether this effect impacts on HIF1α expression. To this end, ovary carcinoma cells were cultured for 24 h without and with PlGF (10 ng/ml). Orai1, STIM1 and HIF1α transcript levels were quantified utilizing RT-PCR and Orai1, STIM1 and HIF1α protein levels by Western blotting. [Ca] was estimated from Fura-2-fluorescence and SOCE from increase of [Ca] following Ca re-addition after Ca-store depletion with extracellular Ca removal and sarcoendoplasmatic Ca-ATPase (SERCA) inhibitor thapsigargin (1 μM). As a result, exposure of ovary carcinoma cells to PlGF was followed by a significant increase of Orai1 as well as STIM1 transcript and protein levels. PlGF significantly increased store-operated Ca-entry following re-addition of extracellular Ca, an effect virtually abrogated by Orai1 inhibitor MRS1845 (10 μM). PlGF further increased HIF1α transcript and protein levels, an effect again significantly blunted by MRS1845 (10 μM). In conclusion, PlGF upregulates expression of both, Orai1 and STIM1 thus enhancing store-operated Ca-entry with subsequent upregulation of HIF1α.
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http://dx.doi.org/10.1016/j.bbrc.2019.03.025DOI Listing
May 2019

Upregulation of Orai1 and STIM1 expression as well as store-operated Ca entry in ovary carcinoma cells by placental growth factor.

Biochem Biophys Res Commun 2019 05 20;512(3):467-472. Epub 2019 Mar 20.

Department of Internal Medicine III, Eberhard Karls,University, Tübingen, Germany. Electronic address:

Placental growth factor (PlGF) is produced by tumor cells and stimulates tumor growth and metastasis in part by upregulation of hypoxia inducible factor HIF1α. Orchestration of tumor cell proliferation and migration involves oscillations of cytosolic Ca activity ([Ca]). The [Ca] oscillations could be accomplished by triggering of intracellular Ca release followed by store-operated Ca-entry (SOCE). Mechanisms accomplishing SOCE include the pore-forming ion channel unit Orai1 and its regulator STIM1. The present study explored whether PlGF influences the expression of Orai1 and STIM1, as well as SOCE and whether this effect impacts on HIF1α expression. To this end, ovary carcinoma cells were cultured for 24 h without and with PlGF (10 ng/ml). Orai1, STIM1 and HIF1α transcript levels were quantified utilizing RT-PCR and Orai1, STIM1 and HIF1α protein levels by Western blotting. [Ca] was estimated from Fura-2-fluorescence and SOCE from increase of [Ca] following Ca re-addition after Ca-store depletion with extracellular Ca removal and sarcoendoplasmatic Ca-ATPase (SERCA) inhibitor thapsigargin (1 μM). As a result, exposure of ovary carcinoma cells to PlGF was followed by a significant increase of Orai1 as well as STIM1 transcript and protein levels. PlGF significantly increased store-operated Ca-entry following re-addition of extracellular Ca, an effect virtually abrogated by Orai1 inhibitor MRS1845 (10 μM). PlGF further increased HIF1α transcript and protein levels, an effect again significantly blunted by MRS1845 (10 μM). In conclusion, PlGF upregulates expression of both, Orai1 and STIM1 thus enhancing store-operated Ca-entry with subsequent upregulation of HIF1α.
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http://dx.doi.org/10.1016/j.bbrc.2019.03.025DOI Listing
May 2019

Genetic deficiency of the tumor suppressor protein p53 influences erythrocyte survival.

Apoptosis 2018 12;23(11-12):641-650

Department of Internal Medicine III, Eberhard-Karls University Tübingen, Tübingen, Germany.

The transcription factor p53 suppresses tumor growth by inducing nucleated cell apoptosis and cycle arrest. Because of its influence on primitive erythroid cell differentiation and survival, p53 is an important determinant of erythropoiesis. However, the impact of p53 on the fate of erythrocytes, cells lacking nucleus and mitochondria, during their post-maturation phase in the circulation remained elusive. Erythrocyte survival may be compromised by suicidal erythrocyte death or eryptosis, which is hallmarked by phosphatidylserine translocation and stimulated by increase of cytosolic Ca concentration. Here, we comparatively examined erythrocyte homeostasis in p53-mutant mice (Trp53/J) and in corresponding WT mice (C57BL/6J) by analyzing eryptosis and erythropoiesis. To this end, spontaneous cell membrane phosphatidylserine exposure and cytosolic Ca concentration were higher in erythrocytes drawn from Trp53/J mice than from WT mice. Eryptosis induced by glucose deprivation, a pathophysiological cell stressor, was slightly, but significantly more prominent in erythrocytes drawn from Trp53/J mice as compared to WT mice. The loss of erythrocytes by eryptosis was fully compensated by enhanced erythropoiesis in Trp53/J mice, as reflected by increased reticulocytosis and abundance of erythroid precursor cells in the bone marrow. Accordingly, erythrocyte number, packed cell volume and hemoglobin were similar in Trp53/J and WT mice. Taken together, functional p53 deficiency enhances the turnover of circulating erythrocytes by parallel increase of eryptosis and stimulated compensatory erythropoiesis.
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http://dx.doi.org/10.1007/s10495-018-1481-8DOI Listing
December 2018

Heterotrimeric G-protein subunit Gα contributes to agonist-sensitive apoptosis and degranulation in murine platelets.

Physiol Rep 2018 09;6(17):e13841

Department of Vegetative & Clinical Physiology, Eberhard-Karls University, Tübingen, Germany.

Gα , a heterotrimeric G-protein subunit, regulates various cell functions including ion channel activity, cell differentiation, proliferation and apoptosis. Platelet-expressed Gα is decisive for the extent of tissue injury following ischemia/reperfusion. However, it is not known whether Gα plays a role in the regulation of platelet apoptosis, which is characterized by caspase activation, cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) translocation to the platelet surface. Stimulators of platelet apoptosis include thrombin and collagen-related peptide (CoRP), which are further known to enhance degranulation and activation of α β3-integrin and caspases. Using FACS analysis, we examined the impact of agonist treatment on activation and apoptosis in platelets drawn from mice lacking Gα and their wild-type (WT) littermates. As a result, treatment with either thrombin (0.01 U/mL) or CoRP (2 μg/mL or 5 μg/mL) significantly upregulated PS-exposure and significantly decreased forward scatter, reflecting cell size, in both genotypes. Exposure to CoRP triggered a significant increase in active caspase 3, ceramide formation, surface P-selectin, and α β3-integrin activation. These molecular alterations were significantly less pronounced in Gα -deficient platelets as compared to WT platelets. In conclusion, our data highlight a previously unreported role of Gα signaling in governing platelet activation and apoptosis.
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http://dx.doi.org/10.14814/phy2.13841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125243PMC
September 2018

To die or not to die SGK1-sensitive ORAI/STIM in cell survival.

Cell Calcium 2018 09 3;74:29-34. Epub 2018 May 3.

German Center for Neurodegenerative Diseases, Research Site Tübingen, Germany; Department of Neurology and Hertie Institute for Clinical Brain Research, University of Tübingen, Germany.

The pore forming Ca release activated Ca channel (CRAC) isoforms ORAI1-3 and their regulators STIM1,2 accomplish store operated Ca entry (SOCE). Activation of SOCE may lead to cytosolic Ca oscillations, which in turn support cell proliferation and cell survival. ORAI/STIM and thus SOCE are upregulated by the serum and glucocorticoid inducible kinase SGK1, a kinase under powerful genomic regulation and activated by phosphorylation via the phosphoinositol-3-phosphate pathway. SGK1 enhances ORAI1 abundance partially by phosphorylation of Nedd4-2, an ubiquitin ligase priming the channel protein for degradation. The SGK1-phosphorylated Nedd4-2 binds to the protein 14-3-3 and is thus unable to ubiquinate ORAI1. SGK1 further increases the ORAI1 and STIM1 protein abundance by activating nuclear factor kappa B (NF-κB), a transcription factor upregulating the expression of STIM1 and ORAI1. SGK1-sensitive upregulation of ORAI/STIM and thus SOCE is triggered by a wide variety of hormones and growth factors, as well as several cell stressors including ischemia, radiation, and cell shrinkage. SGK1 dependent upregulation of ORAI/STIM confers survival of tumor cells and thus impacts on growth and therapy resistance of cancer. On the other hand, SGK1-dependent upregulation of ORAI1 and STIM1 may support survival of neurons and impairment of SGK1-dependent ORAI/STIM activity may foster neurodegeneration. Clearly, further experimental effort is needed to define the mechanisms linking SGK1-dependent upregulation of ORAI1 and STIM1 to cell survival and to define the impact of SGK1-dependent upregulation of ORAI1 and STIM1 on malignancy and neurodegenerative disease.
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http://dx.doi.org/10.1016/j.ceca.2018.05.001DOI Listing
September 2018

Therapeutic Interference With Vascular Calcification-Lessons From Klotho-Hypomorphic Mice and Beyond.

Front Endocrinol (Lausanne) 2018 4;9:207. Epub 2018 May 4.

Department of Internal Medicine and Cardiology, Charité-Universität Medizin Berlin, Berlin, Germany.

Medial vascular calcification, a major pathophysiological process associated with cardiovascular disease and mortality, involves osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). In chronic kidney disease (CKD), osteo-/chondrogenic transdifferentiation of VSMCs and, thus, vascular calcification is mainly driven by hyperphosphatemia, resulting from impaired elimination of phosphate by the diseased kidneys. Hyperphosphatemia with subsequent vascular calcification is a hallmark of klotho-hypomorphic mice, which are characterized by rapid development of multiple age-related disorders and early death. In those animals, hyperphosphatemia results from unrestrained formation of 1,25(OH)D with subsequent retention of calcium and phosphate. Analysis of klotho-hypomorphic mice and mice with vitamin D overload uncovered several pathophysiological mechanisms participating in the orchestration of vascular calcification and several therapeutic opportunities to delay or even halt vascular calcification. The present brief review addresses the beneficial effects of bicarbonate, carbonic anhydrase inhibition, magnesium supplementation, mineralocorticoid receptor (MR) blockage, and ammonium salts. The case is made that bicarbonate is mainly effective by decreasing intestinal phosphate absorption, and that carbonic anhydrase inhibition leads to metabolic acidosis, which counteracts calcium-phosphate precipitation and VSMC transdifferentiation. Magnesium supplementation, MR blockage and ammonium salts are mainly effective by interference with osteo-/chondrogenic signaling in VSMCs. It should be pointed out that the, by far, most efficient substances are ammonium salts, which may virtually prevent vascular calcification. Future research will probably uncover further therapeutic options and, most importantly, reveal whether these observations in mice can be translated into treatment of patients suffering from vascular calcification, such as patients with CKD.
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http://dx.doi.org/10.3389/fendo.2018.00207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945862PMC
May 2018

Neurons, Erythrocytes and Beyond -The Diverse Functions of Chorein.

Neurosignals 2017 28;25(1):117-126. Epub 2017 Nov 28.

Department of Biochemistry, University of Crete Medical School, Heraklion, Greece.

Chorea-acanthocytosis (ChAc), a neurodegenerative disease, results from loss-of-function-mutations of the chorein-encoding gene VPS13A. Affected patients suffer from a progressive movement disorder including chorea, parkinsonism, dystonia, tongue protrusion, dysarthria, dysphagia, tongue and lip biting, gait impairment, progressive distal muscle wasting, weakness, epileptic seizures, cognitive impairment, and behavioral changes. Those pathologies may be paralleled by erythrocyte acanthocytosis. Chorein supports activation of phosphoinositide-3-kinase (PI3K)-p85-subunit with subsequent up-regulation of ras-related C3 botulinum toxin substrate 1 (Rac1) activity, p21 protein-activated kinase 1 (PAK1) phosphorylation, and activation of several tyrosine kinases. Chorein sensitive PI3K signaling further leads to stimulation of the serum and glucocorticoid inducible kinase SGK1, which in turn upregulates ORAI1, a Ca2+-channel accomplishing store operated Ca2+-entry (SOCE). The signaling participates in the regulation of cytoskeletal architecture on the one side and cell survival on the other. Compromised cytoskeletal architecture has been shown in chorein deficient erythrocytes, fibroblasts and endothelial cells. Impaired degranulation was observed in chorein deficient PC12 cells and in platelets from ChAc patients. Similarly, decreased ORAI1 expression and SOCE as well as compromised cell survival were seen in fibroblasts and neurons isolated from ChAc patients. ORAI1 expression, SOCE and cell survival can be restored by lithium treatment, an effect disrupted by pharmacological inhibition of SGK1 or ORAI1. Chorein, SGK1, ORAI1 and SOCE further confer survival of tumor cells. In conclusion, much has been learned about the function of chorein and the molecular pathophysiology of chorea-acanthocytosis. Most importantly, a treatment halting or delaying the clinical course of this devastating disease may become available. A controlled clinical study is warranted, in order to explore whether the in vitro observations indeed reflect the in vivo pathology of the disease.
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http://dx.doi.org/10.1159/000485457DOI Listing
March 2019

Epigallocatechin-3-gallate (EGCG) up-regulates miR-15b expression thus attenuating store operated calcium entry (SOCE) into murine CD4 T cells and human leukaemic T cell lymphoblasts.

Oncotarget 2017 Oct 8;8(52):89500-89514. Epub 2017 Aug 8.

Department of Internal Medicine III, Tübingen University, Gmelinstraβe, Tübingen, Germany.

CD4 T cells are key elements in immune responses and inflammation. Activation of T cell receptors in CD4 T cells triggers cytosolic Ca release with subsequent store operated Ca entry (SOCE), which is accomplished by the pore forming Ca release activated Ca (CRAC) channel Orai1 and its regulator stromal cell-interaction molecule 2 (STIM2). Green tea polyphenol epigallocatechin-3-gallate (EGCG) acts as a potent anti-inflammatory and anti-oxidant agent for various types of cells including immune cells. However, how post-transcriptional gene regulators such as miRNAs are involved in the regulation of Ca influx into murine CD4 T cells and human Jurkat T cells through EGCG is not defined. EGCG treatment of murine CD4 T cells significantly down-regulated the expression of STIM2 and Orai1 both at mRNA and protein levels. Furthermore, EGCG significantly decreased SOCE in both murine and human T cells. EGCG treatment increased miRNA-15b (miR-15b) abundance in both murine and human T cells. Bioinformatics analysis reveals that miR-15b, which has a STIM2 binding site, is involved in the down-regulation of SOCE. Overexpression of miR-15b significantly decreased the mRNA and protein expression of STIM2 and Orai1 in murine T cells. Treatment of Jurkat T cells with 10 μM EGCG further decreased mTOR and PTEN protein levels. EGCG decreased mitochondrial membrane potential (MMP) in both human and murine T cells. In conclusion, the observations suggest that EGCG inhibits the Ca entry into murine and human T cells, an effect accomplished at least in part by up-regulation of miR-15b.
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http://dx.doi.org/10.18632/oncotarget.20032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685687PMC
October 2017

P38 Kinase, SGK1 and NF-κB Dependent Up-Regulation of Na+/Ca2+ Exchanger Expression and Activity Following TGFß1 Treatment of Megakaryocytes.

Cell Physiol Biochem 2017 15;42(6):2169-2181. Epub 2017 Aug 15.

Department of Molecular Medicine II, Heinrich Heine University Duesseldorf, Duesseldorf, Germany.

Background: TGFβ1, a decisive regulator of megakaryocyte maturation and platelet formation, has previously been shown to up-regulate both, store operated Ca2+ entry (SOCE) and Ca2+ extrusion by Na+/Ca2+ exchange. The growth factor thus augments the increase of cytosolic Ca2+ activity ([Ca2+]i) following release of Ca2+ from intracellular stores and accelerates the subsequent decline of [Ca2+]i. The effect on SOCE is dependent on a signaling cascade including p38 kinase, serum & glucocorticoid inducible kinase SGK1, and nuclear factor NFκB. The specific Na+/Ca2+ exchanger isoforms involved and the signalling regulating the Na+/Ca2+ exchangers remained, however elusive. The present study explored, whether TGFβ1 influences the expression and function of K+ insensitive (NCX) and K+ sensitive (NCKX) Na+/Ca2+ exchangers, and aimed to shed light on the signalling involved.

Methods: In human megakaryocytic cells (MEG01) RT-PCR was performed to quantify NCX/NCKX isoform transcript levels, [Ca2+]i was determined by Fura-2 fluorescence, and Na+/Ca2+ exchanger activity was estimated from the increase of [Ca2+]i following switch from an extracellular solution with 130 or 90 mM Na+ and 0 mM Ca2+ to an extracellular solution with 0 Na+ and 2 mM Ca2+. K+ concentration was 0 mM for analysis of NCX and 40 mM for analysis of NCKX.

Results: TGFβ1 (60 ng/ml, 24 h) significantly increased the transcript levels of NCX1, NCKX1, NCKX2 and NCKX5. Moreover, TGFβ1 (60 ng/ml, 24 h) significantly increased the activity of both, NCX and NCKX. The effect of TGFβ1 on NCX and NCKX transcript levels and activity was significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), the effect on NCX and NCKX activity further by SGK1 inhibitor GSK-650394 (10 µM) and NFκB inhibitor Wogonin (100 µM).

Conclusions: TGFβ1 markedly up-regulates transcription of NCX1, NCKX1, NCKX2, and NCKX5 and thus Na+/Ca2+ exchanger activity, an effect requiring p38 kinase, SGK1 and NFκB.
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http://dx.doi.org/10.1159/000479992DOI Listing
November 2017

Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients.

Cell Physiol Biochem 2017 11;42(5):2066-2077. Epub 2017 Aug 11.

Department of Molecular Medicine II, Heinrich Heine University Duesseldorf, Duesseldorf, Germany.

Background: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment.

Methods: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry.

Results: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB.

Conclusions: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment.
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http://dx.doi.org/10.1159/000479901DOI Listing
November 2017

Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients.

Cell Physiol Biochem 2017 11;42(5):2066-2077. Epub 2017 Aug 11.

Department of Molecular Medicine II, Heinrich Heine University Duesseldorf, Duesseldorf, Germany.

Background: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment.

Methods: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry.

Results: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB.

Conclusions: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment.
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http://dx.doi.org/10.1159/000479901DOI Listing
November 2017

Istaroxime Inhibits Motility and Down-Regulates Orai1 Expression, SOCE and FAK Phosphorylation in Prostate Cancer Cells.

Cell Physiol Biochem 2017 14;42(4):1366-1376. Epub 2017 Jul 14.

Department of Biochemistry, University of Crete Medical School, Voutes, Heraklion, Greece.

Background/aims: Istaroxime is a validated inotropic Na+/K+ ATPase inhibitor currently in development for the treatment of various cardiac conditions. Recent findings established that this steroidal drug exhibits potent apoptotic responses in prostate tumors in vitro and in vivo, by affecting key signaling orchestrating proliferation and apoptosis, such as c-Myc and caspase 3, Rho GTPases and actin cytoskeleton dynamics. In the present study we examined whether istaroxime is affecting cell motility and analyzed the underlying mechanism in prostate tumor cells.

Methods: Migration was assessed by transwell and wound healing assays, Orai1 and Stim1 abundance by RT-PCR and confocal immunofluorescence microscopy, Fura-2 fluorescence was utilized to determine intracellular Ca2+ and Western blotting for FAK/pFAK measurements.

Results: We observed strong inhibition of cell migration in istaroxime treated DU-145 prostate cancer cells. Istaroxime further decreased Orai1 and Stim1 transcript levels and downregulated Orai1 protein expression. Moreover, SOCE was significantly decreased upon istaroxime treatment. Furthermore, istaroxime strikingly diminished phosphorylated FAK levels. Interestingly, the efficacy of istaroxime on the inhibition of DU-145 cell migration was further enhanced by blocking Orai1 with 2-APB and FAK with the specific inhibitor PF-00562271. These results provide strong evidence that istaroxime prevents cell migration and motility of DU-145 prostate tumor cells, an effect at least partially attributed to Orai1 downregulation and FAK de-activation.

Conclusion: Collectively our results indicate that this enzyme inhibitor, besides its pro-apoptotic action, affects motility of cancer cells, supporting its potential role as a strong candidate for further clinical cancer drug development.
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http://dx.doi.org/10.1159/000479200DOI Listing
October 2017

Role of Na+/Ca2+ Exchangers in Therapy Resistance of Medulloblastoma Cells.

Cell Physiol Biochem 2017 3;42(3):1240-1251. Epub 2017 Jul 3.

Department of Molecular Medicine II, Medical Faculty, Heinrich Heine University, Duesseldorf, Germany.

Background/aims: Alterations of cytosolic Ca2+-activity ([Ca2+]i) are decisive in the regulation of tumor cell proliferation, migration and survival. Transport processes participating in the regulation of [Ca2+]i include Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether medulloblastoma cells express Na+/Ca2+-exchangers, whether expression differs between therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells, and whether Na+/Ca2+-exchangers participate in the regulation of cell survival.

Methods: In therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells transcript levels were estimated by RT-PCR, protein abundance by Western blotting, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/ Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free by Na+ free and Ca2+ containing (2 mM) extracellular perfusate as well as cell death from PI -staining and annexin-V binding in flow cytometry.

Results: The transcript levels of NCX3, NCKX2, and NCKX5, protein abundance of NCX3, slope and peak of Δ[Ca2+]i as well as Ica were significantly lower in therapy sensitive D283 than in therapy resistant UW228-3 medulloblastoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i, and augmented the ionizing radiation-induced apoptosis but did not significantly modify clonogenicity of medulloblastoma cells. Apoptosis was further enhanced by NCX3 silencing.

Conclusions: Na+/Ca2+-exchanger activity significantly counteracts apoptosis but does not significantly affect clonogenicity after radiation of medulloblastoma cells.
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http://dx.doi.org/10.1159/000478953DOI Listing
November 2017

NFAT5-sensitive Orai1 expression and store-operated Ca entry in megakaryocytes.

FASEB J 2017 08 26;31(8):3439-3448. Epub 2017 Apr 26.

Department of Cardiology and Vascular Medicine and Physiology, University of Tübingen, Tübingen, Germany;

The transcription factor nuclear factor of activated T cells 5 (NFAT5) is up-regulated in several clinical disorders, including dehydration. NFAT5-sensitive genes include serum and glucocorticoid-inducible kinase 1 (SGK1). The kinase is a powerful regulator of Orai1, a Ca channel accomplishing store-operated Ca entry (SOCE). Orai1 is stimulated after intracellular store depletion by the Ca sensors stromal interaction molecule 1 (STIM1), or STIM2, or both. In the present study, we explored whether nuclear factor of activated T cell (NFAT)-5 influences Ca signaling in megakaryocytes. To this end, human megakaryocytic (MEG-01) cells were transfected with NFAT5 or with siNFAT5. Platelets and megakaryocytes were isolated from wild-type mice with either access to water or dehydration by 36 h of water deprivation. Transcript levels were determined with quantitative RT-PCR and protein abundance by Western blot analysis and flow cytometry, cytosolic (intracellular) Ca concentration ([Ca]) by fura-2-fluorescence. SOCE was estimated from the increase of [Ca] following readdition of extracellular Ca after store depletion with thapsigargin (1 µM). Platelet degranulation was estimated from P-selectin abundance and integrin activation from αβ integrin abundance determined by flow cytometry. As a result, NFAT5 transfection or exposure to hypertonicity (+40 mM NaCl) of MEG-01 cells increased Orai1, Orai2, STIM1, and STIM2 transcript levels. Orai1 transcript levels were decreased by NFAT5 silencing. NFAT5 transfection and IκB inhibitor BMS 345541 (5 µM) increased SOCE, whereas NFAT5 silencing and SGK1 inhibitor GSK650394 (10 µM) decreased SOCE. In the mice, dehydration increased NFAT5 and Orai1 protein abundance in megakaryocytes and NFAT5, Orai1, and Orai2 abundance in platelets. Dehydration further augmented the degranulation and integrin activation by thrombin and collagen-related peptide. In summary, NFAT5 is a powerful regulator of Orai1-expression and SOCE in megakaryocytes.-Sahu, I., Pelzl, L., Sukkar, B., Fakhri, H., al-Maghout, T., Cao, H., Hauser, S., Gutti, R., Gawaz, M., Lang, F. NFAT5-sensitive Orai1 expression and store-operated Ca entry in megakaryocytes.
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http://dx.doi.org/10.1096/fj.201601211RDOI Listing
August 2017

Down-Regulation of the Na+,Cl- Coupled Creatine Transporter CreaT (SLC6A8) by Glycogen Synthase Kinase GSK3ß.

Cell Physiol Biochem 2016 15;40(5):1231-1238. Epub 2016 Dec 15.

Department of Physiology I, University of Tuebingen, Tuebingen, Germany.

Background: The Na+,Cl- coupled creatine transporter CreaT (SLC6A8) is expressed in a variety of tissues including the brain. Genetic defects of CreaT lead to mental retardation with seizures. The present study explored the regulation of CreaT by the ubiquitously expressed glycogen synthase kinase GSK3ß, which contributes to the regulation of neuroexcitation. GSK3ß is phosphorylated and thus inhibited by PKB/Akt. Moreover, GSK3ß is inhibited by the antidepressant lithium. The present study thus further tested for the effects of PKB/Akt and of lithium.

Methods: CreaT was expressed in Xenopus laevis oocytes with or without wild-type GSK3ß or inactive K85RGSK3ß. CreaT and GSK3ß were further expressed without and with additional expression of wild type PKB/Akt. Creatine transport in those oocytes was quantified utilizing dual electrode voltage clamp.

Results: Electrogenic creatine transport was observed in CreaT expressing oocytes but not in water-injected oocytes. In CreaT expressing oocytes, co-expression of GSK3ß but not of K85RGSK3ß, resulted in a significant decrease of creatine induced current. Kinetic analysis revealed that GSK3ß significantly decreased the maximal creatine transport rate. Exposure of CreaT and GSK3ß expressing oocytes for 24 hours to Lithium was followed by a significant increase of the creatine induced current. The effect of GSK3ß on CreaT was abolished by co-expression of PKB/Akt.

Conclusion: GSK3ß down-regulates the creatine transporter CreaT, an effect reversed by treatment with the antidepressant Lithium and by co-expression of PKB/Akt.
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http://dx.doi.org/10.1159/000453177DOI Listing
February 2017

Role of Dicer Enzyme in the Regulation of Store Operated Calcium Entry (SOCE) in CD4+ T Cells.

Cell Physiol Biochem 2016 8;39(4):1360-8. Epub 2016 Sep 8.

Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu city, China.

Background/aims: Activation of T cell receptors (TCRs) in CD4+ T cells leads to a cascade of signalling reactions including increase of intracellular calcium (Ca2+) levels with subsequent Ca2+ dependent stimulation of gene expression, proliferation, cell motility and cytokine release. The increase of cytosolic Ca2+ results from intracellular Ca2+ release with subsequent activation of store-operated Ca2+ entry (SOCE). Previous studies suggested miRNAs are required for the development and functions of CD4+ T cells. An enzyme called Dicer is required during the process of manufacturing mature miRNAs from the precursor miRNAs. In this study, we explored whether loss of Dicer in CD4+ T cells affects SOCE and thus Ca2+ dependent regulation of cellular functions.

Methods: We tested the expression of Orai1 by q-RT-PCR and flow cytometry. Further, we measured SOCE by an inverted phase-contrast microscope with the Incident-light fluorescence illumination system using Fura-2. Intracellular Ca2+ was also measured by flow cytometry using Ca2+ sensitive dye Fluo-4.

Results: We found that in Dicer deficient (DicerΔ/Δ) mice Orai1 was downregulated at mRNA and protein level in CD4+ T cells. Further, SOCE was significantly smaller in DicerΔ/Δ CD4+ T cells than in CD4+ T cells isolated from wild-type (Dicerfl/fl) mice.

Conclusion: Our data suggest that miRNAs are required for adequate Ca2+ entry into CD4+ T cells and thus triggering of Ca2+ sensitive immune functions.
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http://dx.doi.org/10.1159/000447840DOI Listing
January 2017

Impact of Na+/Ca2+ Exchangers on Therapy Resistance of Ovary Carcinoma Cells.

Cell Physiol Biochem 2015 17;37(5):1857-68. Epub 2015 Nov 17.

Background/aims: According to previous observations, enhanced store-operated Ca2+-entry (SOCE) accomplished by the pore forming ion channel unit Orai1 and its regulator STIM1 contribute to therapy resistance of ovary carcinoma cells. Ca2+ signaling is further shaped by Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether therapy resistance is further paralleled by altered expression and/or function of Na+/Ca2+-exchangers.

Methods: In therapy resistant (A2780cis) and therapy sensitive (A2780sens) ovary carcinoma cells transcript levels were estimated from RT-PCR, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/Ca2+-exchanger activity from the increase of [Ca2+]i (x0394;[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free extracellular perfusate by Na+ free and Ca2+ containing (2 mM) extracellular perfusate, as well as cell death from PI -staining in flow cytometry.

Results: The transcript levels of NCX3, NCKX4, NCKX5, and NCKX6, slope and peak of x0394;[Ca2+]i as well as Ica were significantly higher in therapy resistant than in therapy sensitive ovary carcinoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted x0394;[Ca2+]i and significantly augmented the cisplatin-induced cell death of therapy resistant ovary carcinoma cells without significantly modifying cisplatin-induced cell death of therapy sensitive ovary carcinoma cells.

Conclusion: Enhanced Na+/Ca2+-exchanger activity may contribute to the therapy sensitivity of ovary carcinoma cells.
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http://dx.doi.org/10.1159/000438547DOI Listing
August 2016

Up-regulation of epithelial Na(+) channel ENaC by human parvovirus B19 capsid protein VP1.

Biochem Biophys Res Commun 2015 Dec 4-11;468(1-2):179-84. Epub 2015 Oct 29.

Department of Physiology, University of Tuebingen, Gmelinstr. 5, 72076 Tuebingen, Germany. Electronic address:

Background: Clinical disorders caused by parvovirus B19 (B19V) infection include endothelial dysfunction with cardiac ischemia. The virus is effective in part by lysophosphatidylcholine-producing phospholipase A2 (PLA2) activity of B19V capsid protein VP1. Mechanisms compromising endothelial function include up-regulation of amiloride sensitive epithelial Na(+)-channel ENaC leading to endothelial cell stiffness. Regulators of ENaC include ubiquitin-ligase Nedd4-2. The present study explored whether VP1 modifies ENaC-activity.

Methods: cRNA encoding ENaC was injected into Xenopus oocytes without or with cRNA encoding VP1. Experiments were made with or without coexpression of Nedd4-2. ENaC activity was estimated from amiloride (50 μM) sensitive current.

Results: Injection of cRNA encoding ENaC into Xenopus oocytes was followed by appearance of amiloride sensitive current, which was significantly enhanced by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding PLA2-negative VP1 mutant (H153A). The effect of VP1 on ENaC was mimicked by treatment of ENaC expressing oocytes with lysophosphatidylcholine (1 μg/ml). The effect of VP1 and lysophosphatidylcholine was not additive. ENaC activity was downregulated by Nedd4-2, an effect not reversed by VP1.

Conclusions: The B19V capsid protein VP1 up-regulates ENaC, an effect at least partially due to phospholipase A2 (PLA) dependent formation of lysophosphatidylcholine.
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http://dx.doi.org/10.1016/j.bbrc.2015.10.137DOI Listing
March 2016
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