48 results on '"Eiseler, Tim"'
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2. A pancreatic cancer organoid-in-matrix platform shows distinct sensitivities to T cell killing
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Lahusen, Anton, Cai, Jierui, Schirmbeck, Reinhold, Wellstein, Anton, Kleger, Alexander, Seufferlein, Thomas, Eiseler, Tim, and Lin, Yuan-Na
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- 2024
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3. Loss of ORP3 induces aneuploidy and promotes bladder cancer cell invasion through deregulated microtubule and actin dynamics
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Wang, Xue, Liu, Junnan, Azoitei, Anca, Eiseler, Tim, Meessen, Sabine, Jiang, Wencheng, Zheng, Xi, Makori, Arika W., Eckstein, Markus, Hartmann, Arndt, Stilgenbauer, Stephan, Elati, Mohamed, Hohwieler, Meike, Kleger, Alexander, John, Axel, Zengerling, Friedemann, Wezel, Felix, Bolenz, Christian, and Günes, Cagatay
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- 2023
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4. Temperature-sensitive migration dynamics in neutrophil-differentiated HL-60 cells
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Khachaturyan, Galina, Holle, Andrew W., Ende, Karen, Frey, Christoph, Schwederski, Heiko A., Eiseler, Tim, Paschke, Stephan, Micoulet, Alexandre, Spatz, Joachim P., and Kemkemer, Ralf
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- 2022
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5. SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas
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Müller, Janis A., Groß, Rüdiger, Conzelmann, Carina, Krüger, Jana, Merle, Uta, Steinhart, Johannes, Weil, Tatjana, Koepke, Lennart, Bozzo, Caterina Prelli, Read, Clarissa, Fois, Giorgio, Eiseler, Tim, Gehrmann, Julia, van Vuuren, Joanne, Wessbecher, Isabel M., Frick, Manfred, Costa, Ivan G., Breunig, Markus, Grüner, Beate, Peters, Lynn, Schuster, Michael, Liebau, Stefan, Seufferlein, Thomas, Stenger, Steffen, Stenzinger, Albrecht, MacDonald, Patrick E., Kirchhoff, Frank, Sparrer, Konstantin M. J., Walther, Paul, Lickert, Heiko, Barth, Thomas F. E., Wagner, Martin, Münch, Jan, Heller, Sandra, and Kleger, Alexander
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- 2021
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6. Emerging Roles of Small Extracellular Vesicles in Gastrointestinal Cancer Research and Therapy.
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Schneider, Nora, Hermann, Patrick Christian, Eiseler, Tim, and Seufferlein, Thomas
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STOMACH tumors ,DISEASE progression ,CELL receptors ,IMMUNOMODULATORS ,METASTASIS ,CELL communication ,CELLULAR signal transduction ,MESSENGER RNA ,CELL lines ,TUMOR markers ,EXTRACELLULAR vesicles ,MEDICAL research - Abstract
Simple Summary: Gastrointestinal cancers originate in the digestive system and harbor distinct characteristics according to their site of origin. This review focusses on the role of small extracellular vesicles (sEVs, exosomes) in the progression, metastasis, and treatment of the main GI cancer entities, such as colorectal cancer, gastric cancer, hepatocellular carcinoma, and pancreatic ductal adenocarcinoma. In recent years, sEVs have gained increasing attention as important mediators of intercellular communication within the local tumor microenvironment, and also to distant metastatic niches. sEVs deliver bioactive cargos, like proteins, mRNA, and miRNAs, to reprogram target cells, promoting tumor growth, invasion, immune suppression, and metastasis to specific organs. Due to their presence in all biological fluids, sEVs are ideal biomarker platforms for multiplexing analysis. Furthermore, sEV engineering generated promising approaches for the use of sEV-based therapeutic nanovesicles in GI cancer treatment. Discovered in the late eighties, sEVs are small extracellular nanovesicles (30–150 nm diameter) that gained increasing attention due to their profound roles in cancer, immunology, and therapeutic approaches. They were initially described as cellular waste bins; however, in recent years, sEVs have become known as important mediators of intercellular communication. They are secreted from cells in substantial amounts and exert their influence on recipient cells by signaling through cell surface receptors or transferring cargos, such as proteins, RNAs, miRNAs, or lipids. A key role of sEVs in cancer is immune modulation, as well as pro-invasive signaling and formation of pre-metastatic niches. sEVs are ideal biomarker platforms, and can be engineered as drug carriers or anti-cancer vaccines. Thus, sEVs further provide novel avenues for cancer diagnosis and treatment. This review will focus on the role of sEVs in GI-oncology and delineate their functions in cancer progression, diagnosis, and therapeutic use. [ABSTRACT FROM AUTHOR]
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- 2024
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7. Tbx3 fosters pancreatic cancer growth by increased angiogenesis and activin/nodal-dependent induction of stemness
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Perkhofer, Lukas, Walter, Karolin, Costa, Ivan G., Carrasco, Maria C. Romero, Eiseler, Tim, Hafner, Susanne, Genze, Felicitas, Zenke, Martin, Bergmann, Wendy, Illing, Anett, Hohwieler, Meike, Köhntop, Ralf, Lin, Qiong, Holzmann, Karl-Heinz, Seufferlein, Thomas, Wagner, Martin, Liebau, Stefan, Hermann, Patrick C., Kleger, Alexander, and Müller, Martin
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- 2016
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8. Comparative panel sequencing of DNA variants in cf-, ev- and tumorDNA for pancreatic ductal adenocarcinoma patients
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Waldenmaier, Mareike, Schulte, Lucas, Schönfelder, Jonathan, Fürstberger, Axel, Kraus, Johann M., Daiss, Nora, Seibold, Tanja, Morawe, Mareen, Ettrich, Thomas J., Kestler, Hans A., Kahlert, Christoph, Seufferlein, Thomas, Eiseler, Tim, and Wong, David
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Cancer Research ,liquid biopsy ,PDAC ,extracellular vesicles ,exosomes ,cfDNA ,next-generation sequencing ,Pancreatic neoplasms ,Liquid biopsy ,Extracellular vesicles ,Exosomes ,DDC 570 / Life sciences ,Oncology ,ddc:570 ,High throughput screening ,Biopsie ,ddc:610 ,Exosom ,Bauchspeicheldrüsenkrebs ,%22">Exosom ,DDC 610 / Medicine & health ,Cell-free nucleic acids ,High-throughput nucleotide sequencing - Abstract
Simple Summary Pancreatic ductal adenocarcinoma (PDAC) has still a dismal prognosis. To improve treatment, personalized medicine uses next-generation DNA sequencing to monitor disease and guide treatment decisions. Tumor samples for sequencing are usually obtained by invasive fine-needle biopsy. Recently, the focus has been increasingly shifting to blood-based liquid biopsies, including circulating free (cf)DNA or DNA isolated from extracellular vesicles (evDNA). To evaluate the detection performance of DNA alterations, we directly compared tumor-, cf- and evDNA from patients with advanced PDAC upon panel sequencing. Copy number variations (CNVs), single nucleotide variants (SNVs) and insertions and deletions (indels) were compared for their concordance with tumorDNA. Compared to cfDNA, evDNA contained significantly larger DNA fragments, which improved the concordance of SNVs and indels with tumorDNA. In line with previous observations, CNV detection was mostly uninformative for cf- and evDNA. However, the combination of both liquid biopsy analytes was clearly superior for SNV detection, pointing to potentially improved actionable variant prediction. Abstract Pancreatic ductal adenocarcinomas (PDACs) are tumors with poor prognosis and limited treatment options. Personalized medicine aims at characterizing actionable DNA variants by next-generation sequencing, thereby improving treatment strategies and outcomes. Fine-needle tumor biopsies are currently the gold standard to acquire samples for DNA profiling. However, liquid biopsies have considerable advantages as they are minimally invasive and frequently obtainable and thus may help to monitor tumor evolution over time. However, which liquid analyte works best for this purpose is currently unclear. Our study aims to directly compare tumor-, circulating free (cf-) and extracellular vesicle-derived (ev)DNA by panel sequencing of matching patient material. We evaluated copy number variations (CNVs), single nucleotide variants (SNVs) and insertions and deletions (indels). Our data show that evDNA contains significantly larger DNA fragments up to 5.5 kb, in line with previous observations. Stringent bioinformatic processing revealed a significant advantage of evDNA with respect to cfDNA concerning detection performance for SNVs and a numerical increase for indels. A combination of ev- and cfDNA was clearly superior for SNV detection, as compared to either single analyte, thus potentially improving actionable variant prediction upon further optimization. Finally, calling of CNVs from liquid biopsies still remained challenging and uninformative., publishedVersion
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- 2022
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9. Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling
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Hohwieler, Meike, Illing, Anett, Hermann, Patrick C, Mayer, Tobias, Stockmann, Marianne, Perkhofer, Lukas, Eiseler, Tim, Antony, Justin S, Müller, Martin, Renz, Susanne, Kuo, Chao-Chung, Lin, Qiong, Sendler, Matthias, Breunig, Markus, Kleiderman, Susanne M, Lechel, André, Zenker, Martin, Leichsenring, Michael, Rosendahl, Jonas, Zenke, Martin, Sainz, Bruno, Jr, Mayerle, Julia, Costa, Ivan G, Seufferlein, Thomas, Kormann, Michael, Wagner, Martin, Liebau, Stefan, and Kleger, Alexander
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- 2017
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10. Endothelial Protein kinase D1 is a major regulator of posttraumatic hyperinflammation.
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Schönfelder, Jonathan, Seibold, Tanja, Morawe, Mareen, Sroka, Robert, Schneider, Nora, Cai, Jierui, Golomejic, Josip, Schütte, Lena, Armacki, Milena, Huber-Lang, Markus, Kalbitz, Miriam, Seufferlein, Thomas, and Eiseler, Tim
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PROTEIN kinases ,INFLAMMATION ,ENDOTHELIAL cells ,CELL communication ,TISSUE adhesions - Abstract
Trauma is a major cause of death worldwide. The post-traumatic immune response culminates in the release of pro-inflammatory mediators, translating in the infiltration of neutrophils (PMNs) at injury sites. The extent of this inflammation is determined by multiple factors, such as PMN adhesion to the endothelium, transendothelial migration, endothelial barrier integrity as well as PMN swarming, mass infiltration and activation. This process is initiated by secondary lipid mediators, such as leukotriene B
4 (LTB4 ). We here provide evidence that Protein kinase D1 (PRKD1) in endothelial cells is implicated in all these processes. Endothelial PRKD1 is activated by pro-inflammatory stimuli and amplifies PMN-mediated inflammation by upregulation of cytokine and chemokines as well as adhesion molecules, such as ICAM-1, VCAM-1 and Eselectin. This induces enhanced PMN adhesion and trans-migration. PRKD1 activation also destabilizes endothelial VE-cadherin adhesion complexes and thus the endothelial barrier, fostering PMN infiltration. We even describe a yet unrecognized PRKD1-dependant mechanism to induce biosynthesis of the PMN-swarming mediator LTB4 directed via intercellular communication through small extracellular vesicles (sEVs) and enhanced CXCL8 secretion from activated endothelial cells. These endothelial sEVs transfer the LTB4 biosynthesis enzyme LTA4 hydrolase (LTA4H) to prime PMNs, while initiating biosynthesis also requires additional signals, like CXCL8. We further demonstrate the respective LTA4H-positive sEVs in the serum of polytrauma patients, peaking 12 h post injury. Therefore, PRKD1 is a key regulator in the coordinated communication of the endothelium with PMNs and a vital signaling node during posttraumatic inflammation. [ABSTRACT FROM AUTHOR]- Published
- 2023
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11. Small extracellular vesicles propagate the inflammatory response after trauma
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Seibold, Tanja, Schönfelder, Jonathan, Weeber, Florian, Lechel, André, Armacki, Milena, Waldenmaier, Mareike, Wille, Christoph, Palmer, Annette, Halbgebauer, Rebecca, Karasu, Ebru, Huber‐Lang, Markus, Kalbitz, Miriam, Radermacher, Peter, Paschke, Stephan, Seufferlein, Thomas, and Eiseler, Tim
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Male ,Neutrophils ,Science ,Lunge ,Small extracellular vesicles ,Shock, Hemorrhagic ,Interleukin 8 ,Exosomes ,Trauma ,Mice ,H��morrhagischer Schock ,Sepsis ,Animals ,ddc:610 ,Endothelium ,%22">Exosom ,Lung ,Research Articles ,NEUTROPHIL MIGRATION ,Inflammation ,Respiratory Distress Syndrome ,IL-8 ,Multiple Trauma ,Hämorrhagischer Schock ,Wounds and injuries ,Interleukin-8 ,ENDOTHELIAL BARRIER FUNCTION ,Endothelial Cells ,PULMONARY CONTUSION ,MOUSE MODEL ,Acute Kidney Injury ,respiratory system ,Extracellular vesicles ,Mice, Inbred C57BL ,Disease Models, Animal ,Neutrophil Infiltration ,CELLS ,Exosom ,Neutrophiler Granulozyt ,DDC 610 / Medicine & health ,Endothel ,Research Article - Abstract
Trauma is the leading cause of death in individuals under 44 years of age. Thorax trauma (TxT) is strongly associated with trauma‐related death, an unbalanced innate immune response, sepsis, acute respiratory distress syndrome, and multiple organ dysfunction. It is shown that different in vivo traumata, such as TxT or an in vitro polytrauma cytokine cocktail trigger secretion of small extracellular nanovesicles (sEVs) from endothelial cells with pro‐inflammatory cargo. These sEVs transfer transcripts for ICAM‐1, VCAM‐1, E‐selectin, and cytokines to systemically activate the endothelium, facilitate neutrophil‐endothelium interactions, and destabilize barrier integrity. Inhibition of sEV‐release after TxT in mice ameliorates local as well as systemic inflammation, neutrophil infiltration, and distant organ damage in kidneys (acute kidney injury, AKI). Vice versa, injection of TxT‐plasma‐sEVs into healthy animals is sufficient to trigger pulmonary and systemic inflammation as well as AKI. Accordingly, increased sEV concentrations and transfer of similar cargos are observed in polytrauma patients, suggesting a fundamental pathophysiological mechanism., It is shown that thorax trauma in mice facilitates enhanced release of small extracellular nanovesicles (sEVs) with proinflammatory features, which originate from endothelial cells. Injection of TxT‐plasma‐sEVs into healthy animals triggers local and systemic inflammation. Mechanistically, endothelial‐derived sEVs transfer transcripts for cytokines and adhesion molecules to propagate the inflammatory response. Similar sEVs are detected in polytrauma patients suggesting a fundamental pathophysiological mechanism.
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- 2021
12. Small extracellular vesicles and metastasis : blame the messenger
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Seibold, Tanja, Waldenmaier, Mareike, Seufferlein, Thomas, and Eiseler, Tim
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Krebs ,pre-metastatic niche ,Immune evasion ,Neoplasm metastasis ,%22">Krebs ,Metastase ,organotropism ,Extracellular vesicles ,Exosomes ,small extracellular vesicles (sEVs) ,Neoplasms ,ddc:610 ,Exosom ,%22">Exosom ,DDC 610 / Medicine & health ,Cancer - Abstract
publishedVersion
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- 2021
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13. Midkine is elevated after multiple trauma and acts directly on human cardiomyocytes by altering their functionality and metabolism
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Lackner, Ina, Weber, Birte, Baur, Meike, Haffner-Luntzer, Melanie, Eiseler, Tim, Fois, Giorgio, Gebhard, Florian, Relja, Borna, Marzi, Ingo, Pfeifer, Roman, Halvachizadeh, Sascha, Lipiski, Miriam, Cesarovic, Nikola, Pape, Hans-Christoph, Kalbitz, Miriam, Auner, Birgit, Störmann, Philipp, Simon, Tim P., Marx, Gernot, Haug, Alexander, Egerer, L., Giensven, M.V., Huber-Lang, Markus, Tolba, René, Reiss, K., Uhlig, Stefan, Horst, Klemens, Teuben, Michel, Almahoud, K., Kalbas, Yannik, Luken, H., and Hildebrand, Frank
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Prevention cardiac injury ,Fracture treatment ,Polytrauma ,Cardiac dysfunction ,Damage associated molecular pattern ,Toll-like receptor ,Toll-like receptor signaling ,CytoSorb® 300 - Abstract
Background and Purpose: Post-traumatic cardiac dysfunction often occurs in multiply injured patients (ISS ≥ 16). Next to direct cardiac injury, post-traumatic cardiac dysfunction is mostly induced by the release of inflammatory biomarkers. One of these is the heparin-binding factor Midkine, which is elevated in humans after fracture, burn injury and traumatic spinal cord injury. Midkine is associated with cardiac pathologies but the exact role of Midkine in the development of those diseases is ambiguous. The systemic profile of Midkine after multiple trauma, its effects on cardiomyocytes and the association with post-traumatic cardiac dysfunction, remain unknown. Experimental Approach: Midkine levels were investigated in blood plasma of multiply injured humans and pigs. Furthermore, human cardiomyocytes (iPS) were cultured in presence/absence of Midkine and analyzed regarding viability, apoptosis, calcium handling, metabolic alterations, and oxidative stress. Finally, the Midkine filtration capacity of the therapeutic blood absorption column CytoSorb ®300 was tested with recombinant Midkine or plasma from multiply injured patients. Key Results: Midkine levels were significantly increased in blood plasma of multiply injured humans and pigs. Midkine acts on human cardiomyocytes, altering their mitochondrial respiration and calcium handling in vitro. CytoSorb®300 filtration reduced Midkine concentration ex vivo and in vitro depending on the dosage. Conclusion and Implications: Midkine is elevated in human and porcine plasma after multiple trauma, affecting the functionality and metabolism of human cardiomyocytes in vitro. Further examinations are required to determine whether the application of CytoSorb®300 filtration in patients after multiple trauma is a promising therapeutic approach to prevent post-traumatic cardiac disfunction., Frontiers in Immunology, 10, ISSN:1664-3224
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- 2019
14. Concerted regulation of actin polymerization during constitutive secretion by cortactin and PKD2.
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Weeber, Florian, Becher, Alexander, Seibold, Tanja, Seufferlein, Thomas, and Eiseler, Tim
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POLYMERIZATION ,SECRETION ,PROTEIN kinases ,DEPHOSPHORYLATION ,MICROFILAMENT proteins - Abstract
Constitutive secretion from the trans-Golgi-network (TGN) is facilitated by a concerted regulation of vesicle biogenesis and fission processes. The protein kinase D family (PKD) has been previously described to enhance vesicle fission by modifying the lipid environment. PKD also phosphorylates the actin regulatory protein cortactin at S298 to impair synergistic actin polymerization. We here report additional functions for PKD2 (also known as PRKD2) and cortactin in the regulation of actin polymerization during the fission of transport carriers from the TGN. Phosphorylation of cortactin at S298 impairs the interaction between WIP (also known as WIPF1) and cortactin. WIP stabilizes the autoinhibited conformation of N-WASP (also known asWASL). This leads to an inhibition of synergistic Arp2/3-complex-dependent actin polymerization at the TGN. PKD2 activity at the TGN is controlled by active CDC42-GTP which directly activates N-WASP, inhibits PKD2 and shifts the balance to non- S298-phosphorylated cortactin, which can in turn sequester WIP from N-WASP. Consequently, synergistic actin polymerization at the TGN and constitutive secretion are enhanced. [ABSTRACT FROM AUTHOR]
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- 2019
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15. PKD regulates actin polymerization, neutrophil deformability, and transendothelial migration in response to fMLP and trauma.
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Wille, Christoph, Eiseler, Tim, Langenberger, Sven‐Thorben, Richter, Julia, Mizuno, Kensaku, Radermacher, Peter, Knippschild, Uwe, Huber‐Lang, Markus, Seufferlein, Thomas, and Paschke, Stephan
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PROTEIN kinase inhibitors ,NEUTROPHILS ,BLOOD cell deformability ,WOUNDS & injuries ,CELL migration ,HEMORRHAGIC shock ,ACTIN ,PHENOTYPES - Abstract
Abstract: Neutrophils are important mediators of the innate immune defense and of the host response to a physical trauma. Because aberrant infiltration of injured sites by neutrophils was shown to cause adverse effects after trauma, we investigated how neutrophil infiltration could be modulated at the cellular level. Our data indicate that protein kinase D (PKD) is a vital regulator of neutrophil transmigration. PKD phosphorylates the Cofilin‐phosphatase Slingshot‐2L (SSH‐2L). SSH‐2L in turn dynamically regulates Cofilin activity and actin polymerization in response to a chemotactic stimulus for neutrophils, for example, fMLP. Here, we show that inhibition of PKD by two specific small molecule inhibitors results in broad, unrestricted activation of Cofilin and strongly increases the F‐actin content of neutrophils even under basal conditions. This phenotype correlates with a significantly impaired neutrophil deformability as determined by optical stretcher analysis. Consequently, inhibition of PKD impaired chemotaxis as shown by reduced extravasation of neutrophils. Consequently, we demonstrate that transendothelial passage of both, neutrophil‐like NB4 cells and primary PMNs recovered from a hemorrhagic shock trauma model was significantly reduced. Thus, inhibition of PKD may represent a promising modulator of the neutrophil response to trauma. [ABSTRACT FROM AUTHOR]
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- 2018
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16. The armadillo protein p0071 controls KIF3 motor transport.
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Becher, Alexander, Eiseler, Tim, Porzner, Marc, Walther, Paul, Keil, René, Bobrovich, Susanne, Hatzfeld, Mechthild, and Seufferlein, Thomas
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CHROMOGRANINS , *NEUROENDOCRINE tumors , *KINESIN - Abstract
We here report a novel function of the armadillo protein p0071 (also known as PKP4) during transport mediated by the KIF3 transport complex. Secretion of chromogranin A and matrix metallopeptidase 9 from pancreatic neuroendocrine tumor cells or pancreatic cancer cells, respectively, was substantially reduced following knockdown of p0071. Vesicle tracking indicated that there was impaired directional persistence of vesicle movement upon p0071 depletion. This suggests a disturbed balance between plus- and minus-end directed microtubule transport in cells lacking p0071. p0071 directly interacts with the KIF3 motor subunit KIF3B. Our data indicate that p0071 also interacts with the kinesin cargo adaptor protein KAP3 (also known as KIFAP3) acting as a stabilizing linker between KIF3B and its KAP3 cargo-binding entity. Thus, p0071 is required for directional vesicle movement and secretion of different KIF3- transported carriers, thereby regulating the transport of intracellular membrane vesicles along microtubules. [ABSTRACT FROM AUTHOR]
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- 2017
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17. Role of protein kinase D (PKD) in migration, invasion and cell adhesion of pancreas ductal adenocarcinoma cells
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Eiseler, Tim
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Krebs , Actin ,Protein Kinase D , PKD , Zellmigration , Invasion , Zelladhaesion ,macromolecular substances ,musculoskeletal system ,Protein Kinase D , PKD , migration , invasion , cell adhesion , Actin - Abstract
The pancreas ductal adenocarcinoma (PDAC) is one of the most common types of cancer, accounting for a large number of cancer related deaths. Rapid tumour metastasis is a major problem in pancreatic cancer, and little is known on the molecular events governing this process, however, features like changes in cell shape, modulation of cell-to-cell adhesion, enhanced cell motility and matrix degrading potential seem to be important. In the literature the Protein kinase D (PKD) family of serine/threonine kinases, which consists of 3 structurally related isoforms, PKD1/PKCμ, PKD2 and PKD3/PKCnu has been implicated in the regulation of some of these processes, yet the molecular mechanisms involved largely remained unclear. That's why this study, using PDAC cell lines, focused on the function of PKD in the metastatic progression of cancer cells. In this work, PKD was found to be strongly expressed and also active in Panc89 cells. Focusing on this cell line, PKD was shown to colocalise with F-Actin and respective markers, indicating active Actin remodeling. For example, Arp3, a member of the Actin-related Arp2/3 protein complex, which is responsible for de novo Actin nucleation and dendritic branching of Actin filaments, colocalised with PKD. Further, PKD colocalised with a subcellular pool of Vinculin at the edge of membrane protrusions, again indicating active Actin turnover, possibly at nascent focal complexes, however PKD was not localised to Vinculin-positive mature focal adhesions to the substratum. In addition, PKD localised with Cortactin, which is enriched within lamellipodia and membrane ruffles. It is implicated in the stabilisation of F-Actin branch points and therefore exhibits important functions at the cortical F-Actin cytoskeleton, which are directly linked to cell migration. Surprisingly, PKD was also found to directly bind to F-Actin in vitro. The binding domain was mapped to 46 amino acids in the N-terminal region of PKD. An alignment of the respective PKD sequence indicated highly conserved motifs, both amongst PKD isoforms and also between human, mouse, and Drosophila species, pointing to a general feature of PKDs. Apart from the binding to F-Actin, PKD and Cortactin also interacted biochemically and PKD was shown to phosphorylate Cortactin in vitro at S298, as well as at additional unspecified sites. Unfortunately an in vivo phosphorylation of Cortactin could not be demonstrated up to now. Employing stable Panc89 cell lines, PKD1 impaired 3D cell migration, while for PKD1KD expressing cells, migration was enhanced. These effects can either be explained by a model implicating Cortactin phosphorylation by PKD in the regulation of F-Actin turnover and rigidity, or by the phosphorylation of yet unknown PKD substrates at the respective F-Actin-rich structures, negatively regulating cell migration. Since mutation of the potential Cortactin phosphorylation site Ser298 to an alanine residue also increased cell migration in stable Panc89 cells, Cortactin might be a potential target of PKD in the regulation of F-Actin dynamics. PKD has also been implicated in the regulation of Ca2+-dependent cell-to-cell adhesion. PKD2KD and, to lesser extent, PKD1KD, strongly enhanced cell-to-cell adhesion of stable Panc89 cells, whereas E-Cadherin expression in the respective parental cell lines was reduced. At least in the case of PKD2KD cells, aggregation was only partially dependent on E-Cadherin, pointing to the expression of additional Cadherin isoforms. In the PKD2 expressing cell line, aggregation was very weak, in some assays even resembling the vector control. This phenotype could be correlated with processed E-Cadherin fragments in the supernatant of PKD2 expressing cells, which have been implicated in the literature with the inhibition of cell-to-cell adhesion of cancer cell lines, thereby also increasing their invasive potential. Initial results demonstrated that the E-Cadherin fragments from the supernatant of stable PKD2 expressing Panc89 cells were processed by a serine-protease, possibly Plasmin or cationic Trypsin. How the PKD2 isoform is implicated in this process remains to be investigated further. Yet, these findings are in line with data obtained from expression profiling experiments with the respective stable Panc89 cells, indicating that the urokinase-like Plasminogen-activator-receptor (PLAUR) is strongly up-regulated in PKD2 expressing cells, triggering the activation of the uPA-Plasminogen-Plasmin-cascade, which has been demonstrated to be involved in the processing of E-Cadherin. During the course of this work novel aspects concerning the role of PKD in cell migration and cellular adhesion were revealed. The localisation of PKD at the F-Actin cytoskeleton and its in vitro binding to F-Actin implicate possible functions for the PKD protein family in the regulation of F-Actin dynamics, which might influence cell adhesion and motility., Pankreaskarzinome zählen zu häufigsten Tumorarten und sie weisen dabei eine der geringsten 5 Jahre Überlebensraten bei Krebserkrankungen auf. Bis heute ist nicht viel über die molekularen Ereignisse bekannt, die mit der Metastasierung von Krebszellen einhergehen, jedoch spielen Prozesse wie Veränderung der Zellstruktur, Modulation der Zell-Zell-Adhäsion, verstärkte Zellmigration und verändertes invasives Verhalten der Krebszellen eine wichtige Rolle. In der Fachliteratur wurde auch die Proteinkinase D (PKD) Familie mit diesen Prozessen in Verbindung gebracht, die genauen molekularen Mechanismen sind jedoch noch unklar. Auf Grund von Homologien innerhalb ihrer Kinase Domaine wurden die drei Isoenzyme der PKD Familie: PKD1/PKCµ, PKD2, PKD3/PKCnu in eine Subklasse der Calcium/Calmodulin-abhängigen Kinasen (CAMK) eingeordnet. Ziel dieser Arbeit war es, die Rolle der Proteinkinase D bei den oben genannten Prozessen in Pankreasductaladenokarzinom (PDAK) Zelllinien näher zu untersuchen. Dazu wurden zunächst mehrere PDAK Linien charakterisiert. In Panc89 Zellen konnte dabei eine starke Expression kinase-aktiver PKD festgestellt werden, weshalb weitere Untersuchungen sich zunächst auf diese Linie konzentrierten. Durch konfokale Immunfluoreszenzmikroskopie konnte eine Kolokalisation zwischen PKD und F-Aktin, sowie mit Arp3, einem Teil des Arp2/3 Proteinkomplexes, der für die de novo Aktin-Nukleation verantwortlich ist, festgestellt werden. PKD wurde außerdem mit Cortactin, einem Marker, der in Lamellopodien und "membran ruffles" angereichert ist, kolokalisiert. Laut Literatur, ist Cortactin für die Stabilisierung von F-Aktin-Verzweigungen im kortikalen Aktin Zytoskelett verantwortlich und übt somit wichtige Funktionen bei der molekularen Kontrolle der Zellmigration aus. Desweiteren konnte auch eine direkte in vitro Bindung von PKD an F-Aktin nachgewiesen werden. Die Aktin-Bindedomäne wurde dabei auf 46 Aminosäuren im N-terminalen Bereich von PKD eingegrenzt. Ein Sequenzvergleich der entsprechenden Region zeigte hoch konservierte Motive zwischen den einzelnen PKD Isoformen und auch bei unterschiedlichen Spezies, was auf eine mögliche konservierte Funktion innerhalb der PKD Familie hindeutet. Außer dieser Bindung von PKD an F-Aktin konnte auch eine direkte Interaktion von PKD und Cortactin nachgewiesen werden. Im in vitro Kinasetest wurde Cortactin von PKD am Serin-Rest 298 und an weiteren, noch unbekannten Aminosäuren phosphoryliert. Eine Phosphorylierung an Ser298 konnte bisher jedoch in vivo nicht nachgewiesen werden. Mit Hilfe von stabil PKD exprimierenden Zelllinien wurde das Migrationsverhalten in 3D Transwell-Migrationsexperimenten untersucht. PKD1 verringerte die gerichtete Zellmigration, während eine Kinase-inaktive Variante (PKD1KD) die Zellmigration verstärkte. Diese Effekte von PKD lassen sich entweder durch ein Modell erklären, in dem die Phosphorylierung von Cortactin bei der Regulation des Aktinumbaus und der Stabilisierung von Aktin-Verzweigungen eine Rolle spielt, oder aber über die Phosphorylierung bisher noch unbekannter Substrate am Aktin Zytoskelett, welche die Zellmigration negative regulieren. Da jedoch die Mutation von Ser298 in einen Alanin-Rest ebenfalls zu gesteigerter Zellmigration führte, ist es möglich, dass Cortactin ein potentielles physiologisches Substrat von PKD bei der Regulation des Aktinumbaus darstellt. Im Rahmen dieser Arbeit wurde auch der Einfluss der unterschiedlichen PKD Isoformen und Kinase-inaktiven (KD) Mutanten auf die Ca2+-abhängige Zell-Adhäsion untersucht. Die Expression einer Kinase-inaktiven PKD2-, und in geringerem Ausmaß auch einer PKD1KD-Mutante, steigerte deutlich die Zell-Zell-Adhäsion der Panc89 Zellen, bei gleichzeitig reduzierter E-Cadherin Expression. Da ein E-Cadherin spezifischer Antikörper bei der PKD2-Linie nur partiell mit der Ca2+--abhängigen Zell-Aggreagtion interferierte, deutet dies auf die zusätzliche Expression von anderen Cadherin Isoformen in Panc89 Zellen hin, welche die gesteigerte Zell-Zell-Adhäsion bewirkt haben könnten. Die Expression von PKD2 in Panc89 Zellen führte zu einer deutlich reduzierten Aggregation. Dieser Phänotyp konnte mit N-terminalen E-Cadherin Fragmenten im Zellkulturüberstand korreliert werden, die von einer Serin-Protease abgespalten werden. Als potentielle Kandidaten konnten Plasmin und kationisches Trypsin ausgemacht werden. Dieses Ergebnis ist in guter Übereinstimmung mit "Microarray-Genprofiling"-Experimenten, bei denen eine verstärkte Expression des "urokinase-like Plasminogen-activator-receptor" Gens (PLAUR) in PKD2 exprimierenden Panc89 Zellen festgestellt werden konnte, was wiederum zu einer Aktivierung der Plasmin-Kaskade und somit auch zu der beobachteten E-Cadherin Prozessierung beitragen koennte.
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- 2006
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18. Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation.
- Author
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Sroka, Robert, Van Lint, Johan, Katz, Sarah-Fee, Schneider, Marlon R., Kleger, Alexander, Paschke, Stephan, Seufferlein, Thomas, and Eiseler, Tim
- Subjects
CADHERINS ,PROTEIN kinases ,PHOSPHORYLATION ,HOMEOSTASIS ,MICROFILAMENT proteins - Abstract
Dynamic regulation of cell--cell adhesion by the coordinated formation and dissolution of E-cadherin-based adherens junctions is crucial for tissue homeostasis. The actin-binding protein cortactin interacts with E-cadherin and enables F-actin accumulation at adherens junctions. Here, we were interested to study the broader functional interactions of cortactin in adhesion complexes. In line with literature, we demonstrate that cortactin binds to E-cadherin, and that a posttranslational modification of cortactin, RhoA-induced phosphorylation by protein kinase D1 (PKD1; also known as PRKD1) at S298, impairs adherens junction assembly and supports their dissolution. Two new S298-phosphorylation-dependent interactions were also identified, namely, that phosphorylation of cortactin decreases its interaction with β-catenin and the actin-binding protein vinculin. In addition, binding of vinculin to β-catenin, as well as linkage of vinculin to F-actin, are also significantly compromised upon phosphorylation of cortactin. Accordingly, we found that regulation of cell--cell adhesion by phosphorylation of cortactin downstream of RhoA and PKD1 is vitally dependent on vinculin-mediated protein interactions. Thus, cortactin, unexpectedly, is an important integration node for the dynamic regulation of protein complexes during breakdown and formation of adherens junctions. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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19. Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion.
- Author
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Eiseler, Tim, Wille, Christoph, Koehler, Conny, Illing, Anett, and Seufferlein, Thomas
- Subjects
- *
METALLOPROTEINASES , *GOLGI apparatus , *PROTEIN kinases , *VESICLES (Cytology) , *ADP-ribosylation , *THREONINE , *PROTEIN transport - Abstract
Vesicle formation and fission are tightly regulated at the trans-Golgi network (TGN) during constitutive secretion. Two major protein families regulate these processes: members of the adenosyl-ribosylation factor family of small G-proteins (ARFs) and the protein kinase D (PKD) family of serine/threonine kinases. The functional relationship between these two key regulators of protein transport from the TGN so far is elusive. We here demonstrate the assembly of a novel functional protein complex at the TGN and its key members: cytosolic PKD2 binds ARF-like GTPase (ARL1) and shuttles ARL1 to the TGN. ARL1, in turn, localizes Arfaptin2 to the TGN. At the TGN, where PKD2 interacts with active ARF1, PKD2, and ARL1 are required for the assembly of a complex comprising of ARF1 and Arfaptin2 leading to secretion of matrix metalloproteinase-2 and -7. In conclusion, our data indicate that PKD2 is a core factor in the formation of this multiprotein complex at the TGN that controls constitutive secretion of matrix metalloproteinase cargo. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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20. PKD is recruited to sites of actin remodelling at the leading edge and negatively regulates cell migration
- Author
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Eiseler, Tim, Schmid, Michael A., Topbas, Fitnat, Pfizenmaier, Klaus, and Hausser, Angelika
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- 2007
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21. Phosphorylation and turnover of paxillin in focal contacts is controlled by force and defines the dynamic state of the adhesion site.
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Qin, Ruifang, Schmid, Heidrun, Münzberg, Christin, Maass, Ulrike, Krndija, Denis, Adler, Guido, Seufferlein, Thomas, Liedert, Astrid, Ignatius, Anita, Oswald, Franz, Eiseler, Tim, and von Wichert, Götz
- Published
- 2015
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22. Protein Kinase D family kinases.
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Wille, Christoph, Seufferlein, Thomas, and Eiseler, Tim
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PROTEIN kinases ,PANCREATIC cancer ,CELL motility ,NEOVASCULARIZATION ,METASTASIS ,CANCER cell proliferation - Abstract
Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lower. In our recent study we report that both kinases control PDAC cell invasive properties in an isoform-specific, but opposing manner. PKD1 selectively mediates anti-migratory/anti-invasive features by preferential regulation of the actin-regulatory Cofilin-phosphatase Slingshot1L (SSH1L). PKD2, on the other hand enhances invasion and angiogenesis of PDAC cells in 3D-ECM cultures and chorioallantois tumor models by stimulating expression and secretion of matrixmetalloproteinase 7 and 9 (MMP7/9). MMP9 also enhances PKD2-mediated tumor angiogenesis releasing extracellular matrix-bound VEGF-A. We thus suggest high PKD2 expression and loss of PKD1 may be beneficial for tumor cells to enhance their matrix-invading abilities. In our recent study we demonstrate for the first time PKD1 and 2 isoformselective effects on pancreatic cancer cell invasion, in-vitro and in-vivo, defining isoform-specific regulation of PKDs as a major future issue. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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23. Protein Kinase D1 Mediates Anchorage-dependent and -independent Growth of Tumor Cells via the Zinc Finger Transcription Factor Snail1.
- Author
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Eiseler, Tim, Köhler, Conny, Nimmagadda, Subbaiah Chary, Jamali, Arsia, Funk, Nancy, Joodi, Golsa, Storz, Peter, and Seufferlein, Thomas
- Subjects
- *
PROTEIN kinases , *CANCER cell growth , *ZINC-finger proteins , *TRANSCRIPTION factors , *HISTONE deacetylase - Abstract
We here identify protein kinase D1 (PKD1) as a major regulator of anchorage-dependent and -independent growth of cancer cells controlled via the transcription factor Snail1. Using FRET, we demonstrate that PKD1, but not PKD2, efficiently interacts with Snail1 in nuclei. PKD1 phosphorylates Snail1 at Ser-11. There was no change in the nucleocytoplasmic distribution of Snail1 using wild type Snail1 and Ser-11 phosphosite mutants in different tumor cells. Regardless of its phosphorylation status or following co-expression of constitutively active PKD, Snail1 was predominantly localized to cell nuclei. We also identify a novel mechanism of PKD1-mediated regulation of Snail1 transcriptional activity in tumor cells. The interaction of the co-repressors histone deacetylases 1 and 2 as well as lysyl oxidase-like protein 3 with Snail1 was impaired when Snail1 was not phosphorylated at Ser-11, which led to reduced Snail1-associated histone deacetylase activity. Additionally, lysyl oxidase-like protein 3 expression was up-regulated by ectopic PKD1 expression, implying a synergistic regulation of Snail1-driven transcription. Ectopic expression of PKD1 also up-regulated proliferation markers such as Cyclin D1 and Ajuba. Accordingly, Snail1 and its phosphorylation at Ser-11 were required and sufficient to control PKD1-mediated anchorage-independent growth and anchorage-dependent proliferation of different tumor cells. In conclusion, our data show that PKD1 is crucial to support growth of tumor cells via Snail1. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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24. Keratin 8 phosphorylation regulates keratin reorganization and migration of epithelial tumor cells.
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Busch, Tobias, Armacki, Milena, Eiseler, Tim, Joodi, Golsa, Temme, Claudia, Jansen, Julia, von Wichert, Götz, Omary, M. Bishr, Spatz, Joachim, and Seufferlein, Thomas
- Subjects
KERATIN ,PHOSPHORYLATION ,CELL migration ,CELL motility ,EPITHELIAL cell tumors ,HETEROCHAIN polymers - Abstract
Cell migration and invasion are largely dependent on the complex organization of the various cytoskeletal components. Whereas the role of actin filaments and microtubules in cell motility is well established, the role of intermediate filaments in this process is incompletely understood. Organization and structure of the keratin cytoskeleton, which consists of heteropolymers of at least one type 1 and one type 2 intermediate filament, are in part regulated by post-translational modifications. In particular, phosphorylation events influence the properties of the keratin network. Sphingosylphosphorylcholine (SPC) is a bioactive lipid with the exceptional ability to change the organization of the keratin cytoskeleton, leading to reorganization of keratin filaments, increased elasticity, and subsequently increased migration of epithelial tumor cells. Here we investigate the signaling pathways that mediate SPC-induced keratin reorganization and the role of keratin phosphorylation in this process. We establish that the MEK-ERK signaling cascade regulates both SPC-induced keratin phosphorylation and reorganization in human pancreatic and gastric cancer cells and identify Ser431 in keratin 8 as the crucial residue whose phosphorylation is required and sufficient to induce keratin reorganization and consequently enhanced migration of human epithelial tumor cells. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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25. Protein Kinase D Regulates RhoA Activity via Rhotekin Phosphorylation.
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Pusapati, Ganesh V., Eiseler, Tim, Rykx, An, Vandoninck, Sandy, Derua, Rita, Waelkens, Etienne, Van Lint, Johan, von Wichert, Götz, and Seufferlein, Thomas
- Subjects
- *
PROTEIN kinases , *PHOSPHORYLATION , *CELL membranes , *G proteins , *NEOVASCULARIZATION - Abstract
The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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26. Protein Kinase D Controls Actin Polymerization and Cell Motility through Phosphorylation of Cortactin.
- Author
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Eiseler, Tim, Hausser, Angelika, De Kimpe, Line, Van Lint, Johan, and Pfizenmaier, Klaus
- Subjects
- *
PROTEIN kinases , *ACTIN , *CARRIER proteins , *POLYMERIZATION , *SERINE , *CELL migration , *CANCER cell motility - Abstract
We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein cortactin and the Arp actin polymerization machinery. PKD phosphorylates cortactin in vitro and in vivo at serine 298 thereby generating a 14-3-3 binding motif. In vitro, a phosphorylation-deficient cortactin-S298A protein accelerated VCA-Arp-cortactin-mediated synergistic actin polymerization and showed reduced F-actin binding, indicative of enhanced turnover of nucleation complexes. In vivo, cortactin co-localized with the nucleation promoting factor WAVE2, essential for lamellipodia extension, in the actin polymerization zone in Heregulin-treated MCF-7 cells. Using a 3-dye FRET-based approach we further demonstrate that WAVE2-Arp and cortactin prominently interact at these structures. Accordingly, cortactin-S298A significantly enhanced lamellipodia extension and directed cell migration. Our data thus unravel a previously unrecognized mechanism by which PKD controls cancer cell motility. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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27. Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot.
- Author
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Eiseler, Tim, Döppler, Heike, Yan, Irene K., Kitatani, Kanae, Mizuno, Kensaku, and Storz, Peter
- Subjects
- *
CANCER cells , *PROTEIN kinases , *PHOSPHATASES , *ACTIN , *SERINE , *CELL migration , *BIOLOGICAL research - Abstract
Dynamic actin remodelling processes at the leading edge of migrating tumour cells are concerted events controlled by a fine-tuned temporal and spatial interplay of kinases and phosphatases. Actin severing is regulated by actin depolymerizing factor (ADF)/cofilin, which regulates stimulus-induced lamellipodia protrusion and directed cell motility. Cofilin is activated by dephosphorylation through phosphatases of the slingshot (SSH) family. SSH activity is strongly increased by its binding to filamentous actin (F-actin); however, other upstream regulators remain unknown. Here we show that in response to RhoA activation, protein kinase D1 (PKD1) phosphorylates the SSH enzyme SSH1L at a serine residue located in its actin-binding motif. This generates a 14-3-3-binding motif and blocks the localization of SSH1L to F-actin-rich structures in the lamellipodium by sequestering it in the cytoplasm. Consequently, expression of constitutively active PKD1 in invasive tumour cells enhanced the phosphorylation of cofilin and effectively blocked the formation of free actin-filament barbed ends and directed cell migration. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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28. Loss of cell-cell contacts induces NF-κB via RhoA-mediated activation of protein kinase D1.
- Author
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Cowell, Catherine F., Yan, Irene K., Eiseler, Tim, Leightner, Amanda C., Döppler, Heike, and Storz, Peter
- Published
- 2009
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29. Pancreatic Cancer Small Extracellular Vesicles (Exosomes): A Tale of Short- and Long-Distance Communication.
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Waldenmaier, Mareike, Seibold, Tanja, Seufferlein, Thomas, and Eiseler, Tim
- Subjects
PANCREATIC tumors ,DISEASE progression ,EXOSOMES ,METASTASIS ,CELL communication ,TUMOR markers - Abstract
Simple Summary: Even today, pancreatic cancer still has a dismal prognosis. It is characterized by a lack of early symptoms and thus late diagnosis as well as early metastasis. The majority of patients suffer from pancreatic ductal adenocarcinoma (PDAC). PDACs communicate extensively with cellular components of their microenvironment, but also with distant metastatic niches to facilitate tumor progression and dissemination. This crosstalk is substantially enabled by small extracellular vesicles (sEVs, exosomes) with a size of 30–150 nm that are released from the tumor cells. sEVs carry bioactive cargos that reprogram target cells to promote tumor growth, migration, metastasis, immune evasion, or chemotherapy resistance. Interestingly, sEVs also carry novel diagnostic, prognostic and potentially also predictive biomarkers. Moreover, engineered sEVs may be utilized as therapeutic agents, improving treatment options. The role of sEVs for PDAC development, progression, diagnosis, prognosis, and treatment is the focus of this review. Even with all recent advances in cancer therapy, pancreatic cancer still has a dismal 5-year survival rate of less than 7%. The most prevalent tumor subtype is pancreatic ductal adenocarcinoma (PDAC). PDACs display an extensive crosstalk with their tumor microenvironment (TME), e.g., pancreatic stellate cells, but also immune cells to regulate tumor growth, immune evasion, and metastasis. In addition to crosstalk in the local TME, PDACs were shown to induce the formation of pre-metastatic niches in different organs. Recent advances have attributed many of these interactions to intercellular communication by small extracellular vesicles (sEVs, exosomes). These nanovesicles are derived of endo-lysosomal structures (multivesicular bodies) with a size range of 30–150 nm. sEVs carry various bioactive cargos, such as proteins, lipids, DNA, mRNA, or miRNAs and act in an autocrine or paracrine fashion to educate recipient cells. In addition to tumor formation, progression, and metastasis, sEVs were described as potent biomarker platforms for diagnosis and prognosis of PDAC. Advances in sEV engineering have further indicated that sEVs might once be used as effective drug carriers. Thus, extensive sEV-based communication and applications as platform for biomarker analysis or vehicles for treatment suggest a major impact of sEVs in future PDAC research. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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30. Protein Kinase D1, Reduced in Human Pancreatic Tumors, Increases Secretion of Small Extracellular Vesicles From Cancer Cells That Promote Metastasis to Lung in Mice.
- Author
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Armacki, Milena, Polaschek, Sandra, Waldenmaier, Mareike, Morawe, Mareen, Ruhland, Claudia, Schmid, Rebecca, Lechel, André, Tharehalli, Umesh, Steup, Christoph, Bektas, Yasin, Li, Hongxia, Kraus, Johann M., Kestler, Hans A., Kruger, Stephan, Ormanns, Steffen, Walther, Paul, Eiseler, Tim, and Seufferlein, Thomas
- Abstract
Pancreatic tumor cells release small extracellular vesicles (sEVs, exosomes) that contain lipids and proteins, RNA, and DNA molecules that might promote formation of metastases. It is not clear what cargo these vesicles contain and how they are released. Protein kinase D1 (PRKD1) inhibits cell motility and is believed to be dysregulated in pancreatic ductal adenocarcinomas. We investigated whether it regulates production of sEVs in pancreatic cancer cells and their ability to form premetastatic niches for pancreatic cancer cells in mice. We analyzed data from UALCAN and human pancreatic tissue microarrays to compare levels of PRKD1 between tumor and nontumor tissues. We studied mice with pancreas-specific disruption of Prkd1 (PRKD1
KO mice), mice that express oncogenic KRAS (KC mice), and KC mice with disruption of Prkd1 (PRKD1KO -KC mice). Subcutaneous xenograft tumors were grown in NSG mice from Panc1 cells; some mice were then given injections of sEVs. Pancreata and lung tissues from mice were analyzed by histology, immunohistochemistry, and/or quantitative polymerase chain reaction; we performed nanoparticle tracking analysis of plasma sEVs. The Prkd 1 gene was disrupted in Panc1 cells using CRISPR-Cas9 or knocked down with small hairpin RNAs, or PRKD1 activity was inhibited with the selective inhibitor CRT0066101. Pancreatic cancer cell lines were analyzed by gene-expression microarray, quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. sEVs secreted by Panc1 cell lines were analyzed by flow cytometry, transmission electron microscopy, and mass spectrometry. Levels of PRKD1 were reduced in human pancreatic ductal adenocarcinoma tissues compared with nontumor tissues. PRKD1KO -KC mice developed more pancreatic intraepithelial neoplasia, at a faster rate, than KC mice, and had more lung metastases and significantly shorter average survival time. Serum from PRKD1KO -KC mice had increased levels of sEVs compared with KC mice. Pancreatic cancer cells with loss or inhibition of PRKD1 increased secretion of sEVs; loss of PRKD1 reduced phosphorylation of its substrate, cortactin, resulting in increased F-actin levels at the plasma membrane. sEVs from cells with loss or reduced expression of PRKD1 had altered content, and injection of these sEVs into mice increased metastasis of xenograft tumors to lung, compared with sEVs from pancreatic cells that expressed PRKD1. PRKD1-deficient pancreatic cancer cells showed increased loading of integrin α6β4 into sEVs—a process that required CD82. Human pancreatic ductal adenocarcinoma has reduced levels of PRKD1 compared with nontumor pancreatic tissues. Loss of PRKD1 results in reduced phosphorylation of cortactin in pancreatic cancer cell lines, resulting in increased in F-actin at the plasma membrane and increased release of sEVs, with altered content. These sEVs promote metastasis of xenograft and pancreatic tumors to lung in mice. [ABSTRACT FROM AUTHOR]- Published
- 2020
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31. A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells.
- Author
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Müller, Martin, Schröer, Jana, Azoitei, Ninel, Eiseler, Tim, Bergmann, Wendy, Köhntop, Ralf, Weidgang, Clair, Seufferlein, Thomas, Kleger, Alexander, Lin, Qiong, Zenke, Martin, Costa, Ivan G, Genze, Felicitas, and Liebau, Stefan
- Subjects
PROTEIN kinases ,ISOENZYMES ,PHOSPHOLIPASE D ,BIOLOGICAL systems ,NEOVASCULARIZATION inhibitors - Abstract
The protein kinase D isoenzymes PKD1/2/3 are prominent downstream targets of PKCs (Protein Kinase Cs) and phospholipase D in various biological systems. Recently, we identified PKD isoforms as novel mediators of tumour cell-endothelial cell communication, tumour cell motility and metastasis. Although PKD isoforms have been implicated in physiological/tumour angiogenesis, a role of PKDs during embryonic development, vasculogenesis and angiogenesis still remains elusive. We investigated the role of PKDs in germ layer segregation and subsequent vasculogenesis and angiogenesis using mouse embryonic stem cells (ESCs). We show that mouse ESCs predominantly express PKD2 followed by PKD3 while PKD1 displays negligible levels. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated at the time of germ layer segregation. Time-restricted PKD2-activation limits mesendoderm formation and subsequent cardiovasculogenesis during early differentiation while leading to branching angiogenesis during late differentiation. In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer. Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs. Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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32. Neuregulin Mediates F-actin-driven Cell Migration through Inhibition of Protein Kinase D1 via Rac1 Protein.
- Author
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Döppler, Heike, Bastea, Ligia I., Eiseler, Tim, and Storz, Peter
- Subjects
- *
NEUREGULINS , *BREAST cancer , *CANCER invasiveness , *CELL migration , *PHOSPHORYLATION , *TUMORS - Abstract
Neuregulin (NRG; heregulin) is overexpressed in ∼30% of breast cancers and mediates various processes involved in tumor progression, including tumor cell migration and invasion. Here, we show that NRG mediates its effects on tumor cell migration via PKD1. Downstream of RhoA, PKD1 can prevent directed cell migration through phosphorylation of its substrate SSH1L. NRG exerts its inhibitory effects on PKD1 through Rac1/NADPH oxidase, leading to decreased PKD1 activation loop phosphorylation and decreased activity toward SSH1L. The consequence of PKD1 inhibition by NRG is decreased binding of 14-3-3 to SSH1L, localization of SSH1L to F-actin at the leading edge, and increased cofilin activity, resulting in increased reorganization of the actin cytoskeleton and cell motility. Our data provide a mechanism through which the Rho GTPase Rac1 cross-talks with PKD1 signaling pathways to facilitate directed cell migration. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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33. Thirty-eight-negative kinase 1 mediates trauma-induced intestinal injury and multi-organ failure.
- Author
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Armacki, Milena, Trugenberger, Anna Katharina, Ellwanger, Ann K., Eiseler, Tim, Schwerdt, Christiane, Bettac, Lucas, Langgartner, Dominik, Azoitei, Ninel, Halbgebauer, Rebecca, Groß, Rüdiger, Barth, Tabea, Lechel, André, Walter, Benjamin M., Kraus, Johann M., Wiegreffe, Christoph, Grimm, Johannes, Scheffold, Annika, Schneider, Marlon R., Peuker, Kenneth, and Zeißig, Sebastian
- Subjects
- *
EPITHELIAL cells , *APOPTOSIS , *SYSTEMIC inflammatory response syndrome , *GASTROINTESTINAL diseases , *KINASES , *COLITIS - Abstract
Dysregulated intestinal epithelial apoptosis initiates gut injury, alters the intestinal barrier, and can facilitate bacterial translocation leading to a systemic inflammatory response syndrome (SIRS) and/or multi-organ dysfunction syndrome (MODS). A variety of gastrointestinal disorders, including inflammatory bowel disease, have been linked to intestinal apoptosis. Similarly, intestinal hyperpermeability and gut failure occur in critically ill patients, putting the gut at the center of SIRS pathology. Regulation of apoptosis and immune-modulatory functions have been ascribed to Thirty-eight-negative kinase 1 (TNK1), whose activity is regulated merely by expression. We investigated the effect of TNK1 on intestinal integrity and its role in MODS. TNK1 expression induced crypt-specific apoptosis, leading to bacterial translocation, subsequent septic shock, and early death. Mechanistically, TNK1 expression in vivo resulted in STAT3 phosphorylation, nuclear translocation of p65, and release of IL-6 and TNF-α. A TNF-α neutralizing antibody partially blocked development of intestinal damage. Conversely, gut-specific deletion of TNK1 protected the intestinal mucosa from experimental colitis and prevented cytokine release in the gut. Finally, TNK1 was found to be deregulated in the gut in murine and porcine trauma models and human inflammatory bowel disease. Thus, TNK1 might be a target during MODS to prevent damage in several organs, notably the gut. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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34. Small extracellular vesicles transfer mRNAs and miRNAs to disseminate inflammatory responses after trauma
- Author
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Seibold, Tanja, Eiseler, Tim, Barth, Holger, and Kemkemer, Ralf
- Subjects
Inflammation ,Tiermodell ,Wounds and injuries ,Extracellular vesicles ,Exosomes ,Trauma ,Systemic inflammatory response syndrome ,SIRS ,ddc:610 ,Exosom ,%22">Exosom ,DDC 610 / Medicine & health ,Allgemeine Entzündungsreaktion - Abstract
Physical trauma is the most common cause of death in young individuals and accounts for 10 % of deaths world-wide. Although management of severely injured patients gradually improved in the last decades, the development of dysbalanced immune reactions like the Systemic Inflammatory Response Syndrome (SIRS) is still a major threat. SIRS occurs when the beneficial, local immune response evolves into an exaggerated, systemic inflammation entailing organ failure or even multi organ dysfunction syndrome (MODS). However, the exact mechanisms and regulators involved in the development of SIRS still need to be elucidated in more detail in order to contrive new treatment options. In this thesis a crucial role for small extracellular vesicles (sEVs, exosomes) in the transmission of inflammatory signals in response to trauma was established. It was revealed that different types of trauma, such as thorax trauma (TxT), hemorrhagic shock (HS), but also in vitro trauma with a defined polytrauma cocktail (PTC) induced enhanced secretion of sEVs from endothelial cells. Besides quantitative modulations, trauma-derived sEVs displayed altered cargo content when compared to basal sEVs, comprising transcripts for ICAM-1, VCAM-1, E-Selectin and cytokines, but also miR-298 and miR-34. By transferring their pro-inflammatory cargo in vitro and in vivo, sEVs were shown to trigger inflammatory responses, facilitate neutrophil-endothelium interactions and destabilize the endothelial barrier. Moreover, using the in vitro trauma model with PTC, containing cytokines and anaphylatoxins at concentrations detected in the plasma of polytrauma (PT) patients, it was demonstrated that increased sEV secretion was associated with elevated expression of specific Rab-GTPases involved in ESCRT-dependent and -independent sEV biogenesis pathways. In addition, loading of pro-inflammatory mRNAs in sEVs was shown to depend on the interaction with the RNA-binding protein (RBP) WDR1. Consequently, treatment of TxT mice with the small molecule sEV biogenesis inhibitor GW4869 demonstrated therapeutic potential and ameliorated inflammation in response to trauma. In detail, injection of GW4869 10 min after TxT impaired local as well as systemic inflammatory phenotypes, neutrophil infiltration, endothelial barrier destruction and signs of acute kidney injury (AKI). Clinical relevance of this mechanism was further demonstrated by analyzing PT patient-derived sEVs compared to sEVs from healthy volunteers. Here, PT-sEVs evoked fundamentally enhanced transcription of inflammatory targets as well as activation of pathways implicated in exaggerated immune responses. Hence, results obtained in this thesis suggest a fundamental and general pathophysiological role for sEVs in response to trauma.
- Published
- 2022
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35. Endothelial Protein kinase D1 is a major regulator of post-traumatic hyperinflammation.
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Schönfelder J, Seibold T, Morawe M, Sroka R, Schneider N, Cai J, Golomejic J, Schütte L, Armacki M, Huber-Lang M, Kalbitz M, Seufferlein T, and Eiseler T
- Subjects
- Humans, Cell Adhesion physiology, Endothelium, Vascular metabolism, Animals, Endothelial Cells, Inflammation metabolism, Protein Kinases metabolism, Wounds and Injuries
- Abstract
Trauma is a major cause of death worldwide. The post-traumatic immune response culminates in the release of pro-inflammatory mediators, translating in the infiltration of neutrophils (PMNs) at injury sites. The extent of this inflammation is determined by multiple factors, such as PMN adhesion to the endothelium, transendothelial migration, endothelial barrier integrity as well as PMN swarming, mass infiltration and activation. This process is initiated by secondary lipid mediators, such as leukotriene B
4 (LTB4 ). We here provide evidence that Protein kinase D1 (PRKD1) in endothelial cells is implicated in all these processes. Endothelial PRKD1 is activated by pro-inflammatory stimuli and amplifies PMN-mediated inflammation by upregulation of cytokine and chemokines as well as adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin. This induces enhanced PMN adhesion and trans-migration. PRKD1 activation also destabilizes endothelial VE-cadherin adhesion complexes and thus the endothelial barrier, fostering PMN infiltration. We even describe a yet unrecognized PRKD1-dependant mechanism to induce biosynthesis of the PMN-swarming mediator LTB4 directed via intercellular communication through small extracellular vesicles (sEVs) and enhanced CXCL8 secretion from activated endothelial cells. These endothelial sEVs transfer the LTB4 biosynthesis enzyme LTA4 hydrolase (LTA4 H) to prime PMNs, while initiating biosynthesis also requires additional signals, like CXCL8. We further demonstrate the respective LTA4 H-positive sEVs in the serum of polytrauma patients, peaking 12 h post injury. Therefore, PRKD1 is a key regulator in the coordinated communication of the endothelium with PMNs and a vital signaling node during post-traumatic inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schönfelder, Seibold, Morawe, Sroka, Schneider, Cai, Golomejic, Schütte, Armacki, Huber-Lang, Kalbitz, Seufferlein and Eiseler.)- Published
- 2023
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36. A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactor.
- Author
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Jakl V, Ehmele M, Winkelmann M, Ehrenberg S, Eiseler T, Friemert B, Rojewski MT, and Schrezenmeier H
- Abstract
Mesenchymal stromal cells (MSCs) are promising therapeutic candidates in a variety of diseases due to having immunomodulatory and pro-regenerative properties. In recent years, MSC-derived small extracellular vesicles (sEVs) have attracted increasing interest as a possible alternative to conventional cell therapy. However, translational processes of sEVs for clinical applications are still impeded by inconsistencies regarding isolation procedures and culture conditions. We systematically compared different methods for sEV isolation from conditioned media of ex vivo expanded bone marrow-derived MSCs and demonstrated considerable variability of quantity, purity, and characteristics of sEV preparations obtained by these methods. The combination of cross flow filtration with ultracentrifugation for sEV isolation resulted in sEVs with similar properties as compared to isolation by differential centrifugation combined with ultracentrifugation, the latter is still considered as gold standard for sEV isolation. In contrast, sEV isolation by a combination of precipitation with polyethylene glycol and ultracentrifugation as well as cross flow filtration and size exclusion chromatography resulted in sEVs with different characteristics, as shown by surface antigen expression patterns. The MSC culture requires a growth-promoting supplement, such as platelet lysate, which contains sEVs itself. We demonstrated that MSC culture with EV-depleted platelet lysate does not alter MSC characteristics, and conditioned media of such MSC cultures provide sEV preparations enriched for MSC-derived sEVs. The results from the systematic stepwise evaluation of various aspects were combined with culture of MSCs in a hollow fiber bioreactor. This resulted in a strategy using cross flow filtration with subsequent ultracentrifugation for sEV isolation. In conclusion, this workflow provides a semi-automated, efficient, large-scale-applicable, and good manufacturing practice (GMP)-grade approach for the generation of sEVs for clinical use. The use of EV-depleted platelet lysate is an option to further increase the purity of MSC-derived sEVs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jakl, Ehmele, Winkelmann, Ehrenberg, Eiseler, Friemert, Rojewski and Schrezenmeier.)
- Published
- 2023
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37. Comparative Panel Sequencing of DNA Variants in cf-, ev- and tumorDNA for Pancreatic Ductal Adenocarcinoma Patients.
- Author
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Waldenmaier M, Schulte L, Schönfelder J, Fürstberger A, Kraus JM, Daiss N, Seibold T, Morawe M, Ettrich TJ, Kestler HA, Kahlert C, Seufferlein T, and Eiseler T
- Abstract
Pancreatic ductal adenocarcinomas (PDACs) are tumors with poor prognosis and limited treatment options. Personalized medicine aims at characterizing actionable DNA variants by next-generation sequencing, thereby improving treatment strategies and outcomes. Fine-needle tumor biopsies are currently the gold standard to acquire samples for DNA profiling. However, liquid biopsies have considerable advantages as they are minimally invasive and frequently obtainable and thus may help to monitor tumor evolution over time. However, which liquid analyte works best for this purpose is currently unclear. Our study aims to directly compare tumor-, circulating free (cf-) and extracellular vesicle-derived (ev)DNA by panel sequencing of matching patient material. We evaluated copy number variations (CNVs), single nucleotide variants (SNVs) and insertions and deletions (indels). Our data show that evDNA contains significantly larger DNA fragments up to 5.5 kb, in line with previous observations. Stringent bioinformatic processing revealed a significant advantage of evDNA with respect to cfDNA concerning detection performance for SNVs and a numerical increase for indels. A combination of ev- and cfDNA was clearly superior for SNV detection, as compared to either single analyte, thus potentially improving actionable variant prediction upon further optimization. Finally, calling of CNVs from liquid biopsies still remained challenging and uninformative.
- Published
- 2022
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38. Small Extracellular Vesicles Propagate the Inflammatory Response After Trauma.
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Seibold T, Schönfelder J, Weeber F, Lechel A, Armacki M, Waldenmaier M, Wille C, Palmer A, Halbgebauer R, Karasu E, Huber-Lang M, Kalbitz M, Radermacher P, Paschke S, Seufferlein T, and Eiseler T
- Subjects
- Acute Kidney Injury etiology, Acute Kidney Injury immunology, Acute Kidney Injury physiopathology, Animals, Disease Models, Animal, Endothelial Cells physiology, Extracellular Vesicles physiology, Male, Mice, Mice, Inbred C57BL, Multiple Trauma immunology, Neutrophil Infiltration physiology, Respiratory Distress Syndrome etiology, Respiratory Distress Syndrome immunology, Respiratory Distress Syndrome physiopathology, Sepsis etiology, Sepsis immunology, Sepsis physiopathology, Endothelial Cells immunology, Extracellular Vesicles immunology, Inflammation immunology, Inflammation physiopathology, Multiple Trauma complications
- Abstract
Trauma is the leading cause of death in individuals under 44 years of age. Thorax trauma (TxT) is strongly associated with trauma-related death, an unbalanced innate immune response, sepsis, acute respiratory distress syndrome, and multiple organ dysfunction. It is shown that different in vivo traumata, such as TxT or an in vitro polytrauma cytokine cocktail trigger secretion of small extracellular nanovesicles (sEVs) from endothelial cells with pro-inflammatory cargo. These sEVs transfer transcripts for ICAM-1, VCAM-1, E-selectin, and cytokines to systemically activate the endothelium, facilitate neutrophil-endothelium interactions, and destabilize barrier integrity. Inhibition of sEV-release after TxT in mice ameliorates local as well as systemic inflammation, neutrophil infiltration, and distant organ damage in kidneys (acute kidney injury, AKI). Vice versa, injection of TxT-plasma-sEVs into healthy animals is sufficient to trigger pulmonary and systemic inflammation as well as AKI. Accordingly, increased sEV concentrations and transfer of similar cargos are observed in polytrauma patients, suggesting a fundamental pathophysiological mechanism., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)
- Published
- 2021
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39. Small Extracellular Vesicles and Metastasis-Blame the Messenger.
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Seibold T, Waldenmaier M, Seufferlein T, and Eiseler T
- Abstract
Cancer is a complex disease, driven by genetic defects and environmental cues. Systemic dissemination of cancer cells by metastasis is generally associated with poor prognosis and is responsible for more than 90% of cancer deaths. Metastasis is thought to follow a sequence of events, starting with loss of epithelial features, detachment of tumor cells, basement membrane breakdown, migration, intravasation and survival in the circulation. At suitable distant niches, tumor cells reattach, extravasate and establish themselves by proliferating and attracting vascularization to fuel metastatic growth. These processes are facilitated by extensive cross-communication of tumor cells with cells in the primary tumor microenvironment (TME) as well as at distant pre-metastatic niches. A vital part of this communication network are small extracellular vesicles (sEVs, exosomes) with a size of 30-150 nm. Tumor-derived sEVs educate recipient cells with bioactive cargos, such as proteins, and in particular, major nucleic acid classes, to drive tumor growth, cell motility, angiogenesis, immune evasion and formation of pre-metastatic niches. Circulating sEVs are also utilized as biomarker platforms for diagnosis and prognosis. This review discusses how tumor cells facilitate progression through the metastatic cascade by employing sEV-based communication and evaluates their role as biomarkers and vehicles for drug delivery.
- Published
- 2021
- Full Text
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40. Midkine Is Elevated After Multiple Trauma and Acts Directly on Human Cardiomyocytes by Altering Their Functionality and Metabolism.
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Lackner I, Weber B, Baur M, Haffner-Luntzer M, Eiseler T, Fois G, Gebhard F, Relja B, Marzi I, Pfeifer R, Halvachizadeh S, Lipiski M, Cesarovic N, Pape HC, and Kalbitz M
- Subjects
- Animals, Calcium metabolism, Cell Respiration, Cells, Cultured, Femur injuries, Humans, Laparotomy, Liver injuries, Male, Mitochondria physiology, Reactive Oxygen Species metabolism, Shock, Hemorrhagic, Sus scrofa, Thoracic Injuries, Midkine blood, Multiple Trauma blood, Myocytes, Cardiac physiology
- Abstract
Background and Purpose: Post-traumatic cardiac dysfunction often occurs in multiply injured patients (ISS ≥ 16). Next to direct cardiac injury, post-traumatic cardiac dysfunction is mostly induced by the release of inflammatory biomarkers. One of these is the heparin-binding factor Midkine, which is elevated in humans after fracture, burn injury and traumatic spinal cord injury. Midkine is associated with cardiac pathologies but the exact role of Midkine in the development of those diseases is ambiguous. The systemic profile of Midkine after multiple trauma, its effects on cardiomyocytes and the association with post-traumatic cardiac dysfunction, remain unknown. Experimental Approach: Midkine levels were investigated in blood plasma of multiply injured humans and pigs. Furthermore, human cardiomyocytes (iPS) were cultured in presence/absence of Midkine and analyzed regarding viability, apoptosis, calcium handling, metabolic alterations, and oxidative stress. Finally, the Midkine filtration capacity of the therapeutic blood absorption column CytoSorb ®300 was tested with recombinant Midkine or plasma from multiply injured patients. Key Results: Midkine levels were significantly increased in blood plasma of multiply injured humans and pigs. Midkine acts on human cardiomyocytes, altering their mitochondrial respiration and calcium handling in vitro . CytoSorb®300 filtration reduced Midkine concentration ex vivo and in vitro depending on the dosage. Conclusion and Implications: Midkine is elevated in human and porcine plasma after multiple trauma, affecting the functionality and metabolism of human cardiomyocytes in vitro . Further examinations are required to determine whether the application of CytoSorb®300 filtration in patients after multiple trauma is a promising therapeutic approach to prevent post-traumatic cardiac disfunction.
- Published
- 2019
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41. Src-mediated tyrosine phosphorylation of Protein Kinase D2 at focal adhesions regulates cell adhesion.
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Durand N, Bastea LI, Döppler H, Eiseler T, and Storz P
- Subjects
- Cell Movement, Fluorescent Antibody Technique, Immunohistochemistry, Phosphorylation, Protein Kinase D2, rhoA GTP-Binding Protein metabolism, Cell Adhesion, Focal Adhesions, Protein Kinases metabolism, src-Family Kinases metabolism
- Abstract
Dependent on their cellular localization, Protein Kinase D (PKD) enzymes regulate different processes including Golgi transport, cell signaling and response to oxidative stress. The localization of PKD within cells is mediated by interaction with different lipid or protein binding partners. With the example of PKD2, we here show that phosphorylation events can also contribute to localization of subcellular pools of this kinase. Specifically, in the present study, we show that tyrosine phosphorylation of PKD2 at residue Y87 defines its localization to the focal adhesions and leads to activation. This phosphorylation occurs downstream of RhoA signaling and is mediated via Src. Moreover, mutation of this residue blocks PKD2's interaction with Focal Adhesion Kinase (FAK). The presence and regulation of PKD2 at focal adhesions identifies a novel function for this kinase as a modulator of cell adhesion and migration.
- Published
- 2017
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42. A Dynamic Role of TBX3 in the Pluripotency Circuitry.
- Author
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Russell R, Ilg M, Lin Q, Wu G, Lechel A, Bergmann W, Eiseler T, Linta L, Kumar P P, Klingenstein M, Adachi K, Hohwieler M, Sakk O, Raab S, Moon A, Zenke M, Seufferlein T, Schöler HR, Illing A, Liebau S, and Kleger A
- Subjects
- Animals, Cell Proliferation, Cells, Cultured, Cellular Reprogramming, Gene Deletion, Mice, Mouse Embryonic Stem Cells metabolism, T-Box Domain Proteins analysis, T-Box Domain Proteins genetics, Mouse Embryonic Stem Cells cytology, T-Box Domain Proteins metabolism
- Abstract
Pluripotency represents a cell state comprising a fine-tuned pattern of transcription factor activity required for embryonic stem cell (ESC) self-renewal. TBX3 is the earliest expressed member of the T-box transcription factor family and is involved in maintenance and induction of pluripotency. Hence, TBX3 is believed to be a key member of the pluripotency circuitry, with loss of TBX3 coinciding with loss of pluripotency. We report a dynamic expression of TBX3 in vitro and in vivo using genetic reporter tools tracking TBX3 expression in mouse ESCs (mESCs). Low TBX3 levels are associated with reduced pluripotency, resembling the more mature epiblast. Notably, TBX3-low cells maintain the intrinsic capability to switch to a TBX3-high state and vice versa. Additionally, we show TBX3 to be dispensable for induction and maintenance of naive pluripotency as well as for germ cell development. These data highlight novel facets of TBX3 action in mESCs., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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43. Protein kinase D2 induces invasion of pancreatic cancer cells by regulating matrix metalloproteinases.
- Author
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Wille C, Köhler C, Armacki M, Jamali A, Gössele U, Pfizenmaier K, Seufferlein T, and Eiseler T
- Subjects
- Cell Line, Tumor, Cell Movement genetics, Cell Proliferation, Chorioallantoic Membrane cytology, Extracellular Matrix, HEK293 Cells, HeLa Cells, Humans, Matrix Metalloproteinase 7 biosynthesis, Matrix Metalloproteinase 7 genetics, Matrix Metalloproteinase 7 metabolism, Matrix Metalloproteinase 9 biosynthesis, Matrix Metalloproteinase 9 genetics, Matrix Metalloproteinase 9 metabolism, Neoplasm Metastasis genetics, Neovascularization, Pathologic genetics, Protein Kinase D2, RNA Interference, RNA, Small Interfering, Vascular Endothelial Growth Factor A biosynthesis, Vascular Endothelial Growth Factor A metabolism, Carcinoma, Pancreatic Ductal pathology, Neoplasm Invasiveness genetics, Pancreatic Neoplasms pathology, Protein Kinase C genetics, Protein Kinases genetics
- Abstract
Pancreatic cancer cell invasion, metastasis, and angiogenesis are major challenges for the development of novel therapeutic strategies. Protein kinase D (PKD) isoforms are involved in controlling tumor cell motility, angiogenesis, and metastasis. In particular PKD2 expression is up-regulated in pancreatic cancer, whereas PKD1 expression is lowered. We report that both kinases control pancreatic cancer cell invasive properties in an isoform-specific manner. PKD2 enhances invasion in three-dimensional extracellular matrix (3D-ECM) cultures by stimulating expression and secretion of matrix metalloproteinases 7 and 9 (MMP7/9), by which MMP7 is likely to act upstream of MMP9. Knockdown of MMP7/9 blocks PKD2-mediated invasion in 3D-ECM assays and in vivo using tumors growing on chorioallantois membranes. Furthermore, MMP9 enhances PKD2-mediated tumor angiogenesis by releasing extracellular matrix-bound vascular endothelial growth factor A, increasing its bioavailability and angiogenesis. Of interest, specific knockdown of PKD1 in PKD2-expressing pancreatic cancer cells further enhanced the invasive properties in 3D-ECM systems by generating a high-motility phenotype. Loss of PKD1 thus may be beneficial for tumor cells to enhance their matrix-invading abilities. In conclusion, we define for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion and angiogenesis, in vitro and in vivo, addressing PKD isoform specificity as a major factor for future therapeutic strategies.
- Published
- 2014
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44. A novel protein kinase D phosphorylation site in the tumor suppressor Rab interactor 1 is critical for coordination of cell migration.
- Author
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Ziegler S, Eiseler T, Scholz RP, Beck A, Link G, and Hausser A
- Subjects
- Actins metabolism, Amino Acid Motifs, Amino Acid Sequence, Antibodies immunology, HEK293 Cells, Humans, Molecular Sequence Data, Phosphorylation, Phosphoserine metabolism, Protein Transport, Protein-Tyrosine Kinases metabolism, Subcellular Fractions metabolism, Tumor Suppressor Proteins metabolism, Cell Movement, Intracellular Signaling Peptides and Proteins chemistry, Intracellular Signaling Peptides and Proteins metabolism, Protein Kinase C metabolism
- Abstract
The multifunctional signal adapter protein Ras and Rab interactor 1 (RIN1) is a Ras effector protein involved in the regulation of epithelial cell processes such as cell migration and endocytosis. RIN1 signals via two downstream pathways, namely the activation of Rab5 and Abl family kinases. Protein kinase D (PKD) phosphorylates RIN1 at serine 351 in vitro, thereby regulating interaction with 14-3-3 proteins. Here, we report the identification of serine 292 in RIN1 as an in vivo PKD phosphorylation site. PKD-mediated phosphorylation at this site was confirmed with a phospho-specific antibody and by mass spectrometry. We demonstrate that phosphorylation at serine 292 controls RIN1-mediated inhibition of cell migration by modulating the activation of Abl kinases. We further provide evidence that RIN1 in vivo phosphorylation at serine 351 occurs independently of PKD. Collectively, our data identify a novel PKD signaling pathway through RIN1 and Abl kinases that is involved in the regulation of actin remodeling and cell migration.
- Published
- 2011
- Full Text
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45. Loss of cell-cell contacts induces NF-kappaB via RhoA-mediated activation of protein kinase D1.
- Author
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Cowell CF, Yan IK, Eiseler T, Leightner AC, Döppler H, and Storz P
- Subjects
- Cell Line, Tumor, Epithelial Cells cytology, Epithelial Cells metabolism, Humans, Multiprotein Complexes, Signal Transduction, Cell Communication, NF-kappa B genetics, Protein Kinase C metabolism, Transcriptional Activation, rhoA GTP-Binding Protein metabolism
- Abstract
Cell-cell contacts mediated by cadherins are known to inhibit the small Rho-GTPase RhoA. We here show that in epithelial cells the disruption of these cell-cell contacts as mediated by a calcium switch leads to actin re-organization and the activation of RhoA. We identified the serine/threonine kinase protein kinase D1 (PKD1) as a downstream target for RhoA in this pathway. After disruption of cell-cell contacts, PKD1 relayed RhoA activation to the induction of the transcription factor NF-kappaB. We found that a signaling complex composed of the kinases ROCK, novel protein kinase C (nPKC), and Src family kinases (SFKs) is upstream of PKD1 and crucial for RhoA-mediated NF-kappaB activation. In conclusion, our data suggest a previously undescribed signaling pathway of how RhoA is activated by loss of cell-cell adhesions and by which it mediates the activation of NF-kappaB. We propose that this pathway is of relevance for epithelial tumor cell biology, where loss of cell-cell contacts has been implicated in regulating cell survival and motility.
- Published
- 2009
- Full Text
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46. Protein kinase D1 regulates matrix metalloproteinase expression and inhibits breast cancer cell invasion.
- Author
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Eiseler T, Döppler H, Yan IK, Goodison S, and Storz P
- Subjects
- Blotting, Western, Breast Neoplasms pathology, Carcinoma, Ductal, Breast secondary, Cell Adhesion, Cell Culture Techniques, Cell Line, Tumor, Cell Movement, Cell Proliferation, Gene Expression Regulation, Neoplastic, Humans, Immunoenzyme Techniques, Immunoprecipitation, Lymphatic Metastasis, Neoplasm Invasiveness, RNA, Messenger genetics, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction, Spheroids, Cellular, Breast Neoplasms metabolism, Carcinoma, Ductal, Breast metabolism, Matrix Metalloproteinases metabolism, Protein Kinase C physiology
- Abstract
Introduction: The biological and molecular events that regulate the invasiveness of breast tumour cells need to be further revealed to develop effective therapies that stop breast cancer from expanding and metastasising., Methods: Human tissue samples of invasive breast cancer and normal breast, as well as breast cancer cell lines, were evaluated for protein kinase D (PKD) expression, to test if altered expression could serve as a marker for invasive breast cancer. We further utilised specific PKD1-shRNA and a system to inducibly-express PKD1 to analyse the role of PKD1 in the invasive behaviour of breast cancer cell lines in two-dimensional (2D) and three-dimensional (3D) culture. Invasive behaviour in breast cancer cell lines has been linked to matrix metalloproteinases (MMPs), so we also determined if PKD1 regulates the expression and activity of these enzymes., Results: We found that the serine/threonine kinase, PKD1, is highly expressed in ductal epithelial cells of normal human breast tissue, but is reduced in its expression in more than 95% of all analysed samples of human invasive breast tumours. Additionally, PKD1 is not expressed in highly invasive breast cancer cell lines, whereas non-invasive or very low-invasive breast cancer cell lines express PKD1. Our results further implicate that in MDA-MB-231 cells PKD1 expression is blocked by epigenetic silencing via DNA methylation. The re-expression of constitutively-active PKD1 in MDA-MB-231 cells drastically reduced their ability to invade in 2D and 3D cell culture. Moreover, MCF-7 cells acquired the ability to invade in 2D and 3D cell culture when PKD1 expression was knocked-down by shRNA. PKD1 also regulated the expression of breast cancer cell MMPs, MMP-2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14 and MMP-15, providing a potential mechanism for PKD1 mediation of the invasive phenotype., Conclusions: Our results identify decreased expression of the PKD1 as a marker for invasive breast cancer. They further suggest that the loss of PKD1 expression increases the malignant potential of breast cancer cells. This may be due to the function of PKD1 as a negative regulator of MMP expression. Our data suggest re-expression of PKD1 as a potential therapeutic strategy.
- Published
- 2009
- Full Text
- View/download PDF
47. Protein kinase C(mu) regulation of the JNK pathway is triggered via phosphoinositide-dependent kinase 1 and protein kinase C(epsilon).
- Author
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Brändlin I, Eiseler T, Salowsky R, and Johannes FJ
- Subjects
- 3-Phosphoinositide-Dependent Protein Kinases, Enzyme Activation, Humans, Immunohistochemistry, Isoenzymes genetics, JNK Mitogen-Activated Protein Kinases, Phosphorylation, Protein Kinase C genetics, Protein Kinase C-epsilon, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Tumor Cells, Cultured, Isoenzymes metabolism, MAP Kinase Signaling System physiology, Mitogen-Activated Protein Kinases metabolism, Protein Kinase C metabolism, Protein Serine-Threonine Kinases metabolism
- Abstract
The protein kinase C (PKC)-related enzyme PKC(mu)/PKD (protein kinase D) is activated by activation loop phosphorylation through PKC(eta). Here we demonstrate that PKC(mu) is activated by the direct phosphorylation of PKC(epsilon). PKC(mu) colocalizes with PKC(epsilon) in HEK293 and MCF7 cells as shown by confocal immunofluorescence analyses. PDK1, known as the upstream kinase for several PKC isozymes, associates intracellularly with PKC(epsilon) and PKC(eta). PKC(eta) is phosphorylated by PDK1 in vitro, leading to kinase activation as similarly reported for PKC(epsilon) activation by PDK1. Coexpression of PDK1, PKC(epsilon) and PKC(mu) in HEK293 cells results in PKC(mu) activation. In contrast, the coexpression of PDK1 and PKC(eta) with PKC(mu) does not activate PKC(eta) or consequently PKC(mu). PDK1/PKC(epsilon)-triggered activation of PKC(mu) inhibits JNK, a downstream effector of PKC(mu), whereas upon transient expression of PDK1, PKC(eta), and PKC(mu), JNK is not affected. These data implicate PKC(epsilon) as the biologically important upstream kinase for PKC(mu) in HEK293 cells, regulating downstream effectors. Our results further indicate a PDK1/PKC(eta)/PKC(mu) controlled negative regulation of PKC(eta) kinase activity. In this study, we show that differentially activated kinase cascades involving PDK1 and novel PKC isotypes are responsible for the regulation of PKC(mu) activity and consequently inhibit the JNK pathway.
- Published
- 2002
- Full Text
- View/download PDF
48. Protein kinase C (PKC)eta-mediated PKC mu activation modulates ERK and JNK signal pathways.
- Author
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Brändlin I, Hübner S, Eiseler T, Martinez-Moya M, Horschinek A, Hausser A, Link G, Rupp S, Storz P, Pfizenmaier K, and Johannes FJ
- Subjects
- Breast Neoplasms, Cell Line, DNA Primers, Enzyme Activation, Female, Genes, Reporter, Glutathione Transferase genetics, Green Fluorescent Proteins, Humans, JNK Mitogen-Activated Protein Kinases, Jurkat Cells, Luminescent Proteins genetics, Protein Kinase C genetics, Recombinant Fusion Proteins metabolism, Transfection, Mitogen-Activated Protein Kinases metabolism, Protein Kinase C metabolism
- Abstract
Protein kinase C (PKC), a family of lipid-activated serine kinases, is involved in multiple functions in the regulation of growth control. The PKC-related isoform PKC mu/PKD has been implicated in mitogenic signal cascades because of the activation of p42/p44 MAPK leading to Elk1-mediated gene transcription, and PKC mu/PKD has been shown to be activated via a PKC-dependent pathway. By using confocal analyses, we demonstrate here that PKC mu partially colocalizes with PKC eta in different cell types. Colocalization depends on the presence of the PKC mu pleckstrin homology domain. Coexpression of constitutively active PKC eta with PKC mu leads to a significant enhancement of the PKC mu substrate phosphorylation capacity as a result of an increased phosphorylation of the activation loop Ser(738/742) of PKC mu, whereas Ser(910) autophosphorylation remains unaffected. In vitro phosphorylation experiments show that PKC eta directly phosphorylates PKC mu on activation loop serines. Consequently, the p42 MAPK cascade is triggered leading to an increase in reporter gene activity driven by a serum-responsive element in HEK293 cells. At the same time, PKC eta-mediated JNK activation is reduced, providing evidence for a mutual regulation of PKC mu/PKC eta affecting different arms of the p38/ERK/JNK pathways. Our data provide evidence for the sequential involvement of selective PKC isoforms in kinase cascades and identify the relevant domains in PKC mu for interaction with and activation by PKC eta as pleckstrin homology domain and activation loop.
- Published
- 2002
- Full Text
- View/download PDF
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