Die physiologische Funktion von TRPC6 in Endothelzellen und glatten Muskelzellen der Lunge

Das Ziel der vorliegenden Arbeit war es, die molekularen Mechanismen der hypoxischen pulmonalen Vasokonstriktion in präkapillären pulmonalen arteriellen glatten Muskelzellen (PASMC) sowie des Ischämie-Reperfusionsschadens in den Endothelzellen (LEC) der Säugerlunge zu untersuchen. Da beide fundamentale Mechanismen in TRPC6-defizienten Mäusen nicht mehr auftreten, mussten ihnen eine TRPC6-Aktivierung zugrunde liegen. Zur Aufklärung der Mechanismen auf zellulärer Ebene wurden zuerst PASMC und LEC isoliert und durch Bindung charakteristischer Antikörper identifiziert. Die Identifikation von Signalkomponenten in beiden Zelltypen gelang außerdem durch die Messung der Erhöhung der intrazellulären Ca2+-Konzentration nach Perfusion eines hypoxischen Puffers. Durch Applikation spezifischer pharmakologischer Inhibitoren und Indikatoren gelang es schließlich, Hinweise zum Ablauf der Signaltransduktionskaskaden zu sammeln. In PASMC wird während des sogenannten „priming“ durch eine geringe Rezeptoraktivierung eine basale Konzentration an Diacylglycerin, dem physiologischen Aktivator von TRPC6, gebildet, die jedoch durch die Aktivität von DAG-abbauenden DAG-Kinasen so reduziert wird, dass eine TRPC6-Aktivierung nicht möglich ist. Nach Applikation von Hypoxie führt eine postulierte Erhöhung der reaktiven Sauerstoffradikale in der Zelle jedoch zu einer Inhibition von DAG-Kinasen, zur DAG-Akkumulation und zur TRPC6-Aktivierung. In PASMC werden durch den folgenden Na+-Einstrom spannungsabhängige Ca2+-Kanäle vom L-Typ aktiviert, die den eigentlichen Ca2+-Einstrom zur Zellkontraktion einleiten. In LEC konnte eine ähnliche Signaltransduktionskaskade identifiziert werden, auch wenn hier die Notwendigkeit eines „primings“ nicht geklärt werden konnte und der Ca2+-Einstrom durch TRPC6-Kanäle verläuft, da keine spannungsabhängigen Calciumkanäle vom L-Typ exprimiert werden. Zusammenfassend lässt sich also sagen, dass der TRPC6- Kanal einen wichtigen pharmakologischen Angriffspunkt für beide Signaltransduktionskaskaden darstellt

Caveolin-1 Is a Critical Determinant of Autophagy, Metabolic Switching, and Oxidative Stress in Vascular Endothelium

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules. Caveolin-1null mice have marked metabolic abnormalities, yet the underlying molecular mechanisms are incompletely understood. We found the redox stress plasma biomarker plasma 8-isoprostane was elevated in caveolin-1null mice, and discovered that siRNA-mediated caveolin-1 knockdown in endothelial cells promoted significant increases in intracellular H2O2. Mitochondrial ROS production was increased in endothelial cells after caveolin-1 knockdown; 2-deoxy-D-glucose attenuated this increase, implicating caveolin-1 in control of glycolytic pathways. We performed unbiased metabolomic characterizations of endothelial cell lysates following caveolin-1 knockdown, and discovered strikingly increased levels (up to 30-fold) of cellular dipeptides, consistent with autophagy activation. Metabolomic analyses revealed that caveolin-1 knockdown led to a decrease in glycolytic intermediates, accompanied by an increase in fatty acids, suggesting a metabolic switch. Taken together, these results establish that caveolin-1 plays a central role in regulation of oxidative stress, metabolic switching, and autophagy in the endothelium, and may represent a critical target in cardiovascular diseases

The N Terminus of Adhesion G Protein–Coupled Receptor GPR126/ ADGRG6 as Allosteric Force Integrator

The adhesion G protein–coupled receptor (aGPCR) GPR126/ADGRG6 plays an important role in several physiological functions, such as myelination or peripheral nerve repair. This renders the receptor an attractive pharmacological target. GPR126 is a mechano-sensor that translates the binding of extracellular matrix (ECM) molecules to its N terminus into a metabotropic intracellular signal. To date, the structural requirements and the character of the forces needed for this ECM-mediated receptor activation are largely unknown. In this study, we provide this information by combining classic second-messenger detection with single-cell atomic force microscopy. We established a monoclonal antibody targeting the N terminus to stimulate GPR126 and compared it to the activation through its known ECM ligands, collagen IV and laminin 211. As each ligand uses a distinct mode of action, the N terminus can be regarded as an allosteric module that can fine-tune receptor activation in a context-specific manner

Nitric oxide mediates glial-induced neurodegeneration in Alexander disease

Glia play critical roles in maintaining the structure and function of the nervous system; however, the specific contribution that astroglia make to neurodegeneration in human disease states remains largely undefined. Here we use Alexander disease, a serious degenerative neurological disorder caused by astrocyte dysfunction, to identify glial-derived NO as a signalling molecule triggering astrocyte-mediated neuronal degeneration. We further find that NO acts through cGMP signalling in neurons to promote cell death. Glial cells themselves also degenerate, via the DNA damage response and p53. Our findings thus define a specific mechanism for glial-induced non-cell autonomous neuronal cell death, and identify a potential therapeutic target for reducing cellular toxicity in Alexander disease, and possibly other neurodegenerative disorders with glial dysfunction

Fine Mapping of the 1p36 Deletion Syndrome Identifies Mutation of PRDM16 as a Cause of Cardiomyopathy

Deletion 1p36 syndrome is recognized as the most common terminal deletion syndrome. Here, we describe the loss of a gene within the deletion that is responsible for the cardiomyopathy associated with monosomy 1p36, and we confirm its role in nonsyndromic left ventricular noncompaction cardiomyopathy (LVNC) and dilated cardiomyopathy (DCM). With our own data and publically available data from array comparative genomic hybridization (aCGH), we identified a minimal deletion for the cardiomyopathy associated with 1p36del syndrome that included only the terminal 14 exons of the transcription factor PRDM16 (PR domain containing 16), a gene that had previously been shown to direct brown fat determination and differentiation. Resequencing of PRDM16 in a cohort of 75 nonsyndromic individuals with LVNC detected three mutations, including one truncation mutant, one frameshift null mutation, and a single missense mutant. In addition, in a series of cardiac biopsies from 131 individuals with DCM, we found 5 individuals with 4 previously unreported nonsynonymous variants in the coding region of PRDM16. None of the PRDM16 mutations identified were observed in more than 6,400 controls. PRDM16 has not previously been associated with cardiac disease but is localized in the nuclei of cardiomyocytes throughout murine and human development and in the adult heart. Modeling of PRDM16 haploinsufficiency and a human truncation mutant in zebrafish resulted in both contractile dysfunction and partial uncoupling of cardiomyocytes and also revealed evidence of impaired cardiomyocyte proliferative capacity. In conclusion, mutation of PRDM16 causes the cardiomyopathy in 1p36 deletion syndrome as well as a proportion of nonsyndromic LVNC and DCM

Die physiologische Funktion von TRPC6 in Endothelzellen und glatten Muskelzellen der Lunge

Das Ziel der vorliegenden Arbeit war es, die molekularen Mechanismen der hypoxischen pulmonalen Vasokonstriktion in präkapillären pulmonalen arteriellen glatten Muskelzellen (PASMC) sowie des Ischämie-Reperfusionsschadens in den Endothelzellen (LEC) der Säugerlunge zu untersuchen. Da beide fundamentale Mechanismen in TRPC6-defizienten Mäusen nicht mehr auftreten, mussten ihnen eine TRPC6-Aktivierung zugrunde liegen. Zur Aufklärung der Mechanismen auf zellulärer Ebene wurden zuerst PASMC und LEC isoliert und durch Bindung charakteristischer Antikörper identifiziert. Die Identifikation von Signalkomponenten in beiden Zelltypen gelang außerdem durch die Messung der Erhöhung der intrazellulären Ca2+-Konzentration nach Perfusion eines hypoxischen Puffers. Durch Applikation spezifischer pharmakologischer Inhibitoren und Indikatoren gelang es schließlich, Hinweise zum Ablauf der Signaltransduktionskaskaden zu sammeln. In PASMC wird während des sogenannten „priming“ durch eine geringe Rezeptoraktivierung eine basale Konzentration an Diacylglycerin, dem physiologischen Aktivator von TRPC6, gebildet, die jedoch durch die Aktivität von DAG-abbauenden DAG-Kinasen so reduziert wird, dass eine TRPC6-Aktivierung nicht möglich ist. Nach Applikation von Hypoxie führt eine postulierte Erhöhung der reaktiven Sauerstoffradikale in der Zelle jedoch zu einer Inhibition von DAG-Kinasen, zur DAG-Akkumulation und zur TRPC6-Aktivierung. In PASMC werden durch den folgenden Na+-Einstrom spannungsabhängige Ca2+-Kanäle vom L-Typ aktiviert, die den eigentlichen Ca2+-Einstrom zur Zellkontraktion einleiten. In LEC konnte eine ähnliche Signaltransduktionskaskade identifiziert werden, auch wenn hier die Notwendigkeit eines „primings“ nicht geklärt werden konnte und der Ca2+-Einstrom durch TRPC6-Kanäle verläuft, da keine spannungsabhängigen Calciumkanäle vom L-Typ exprimiert werden. Zusammenfassend lässt sich also sagen, dass der TRPC6- Kanal einen wichtigen pharmakologischen Angriffspunkt für beide Signaltransduktionskaskaden darstellt

Role of PTEN in modulation of ADP-dependent signaling pathways in vascular endothelial cells

ADP plays critical signaling roles in the vascular endothelium. ADP receptors are targeted by several cardiovascular drugs, yet the intracellular pathways modulated by ADP are incompletely understood. These studies have identified important roles for the phosphatase PTEN in ADP-dependent modulation of the endothelial isoform of nitric oxide synthase (eNOS) as well as of lipid and protein kinase pathways in endothelial cells. We find that ADP-promoted eNOS activation as well as phosphorylation of p38 MAPK are enhanced by siRNA-mediated PTEN knockdown. However, the increase in ADP-dependent eNOS activation promoted by PTEN knockdown is abrogated by siRNA-mediated knockdown of p38 MAPK. These findings indicate that PTEN tonically suppresses both p38 phosphorylation as well as ADP-stimulated eNOS activity. A key enzymatic activity of PTEN is its role as a lipid phosphatase, catalyzing the dephosphorylation of phosphoinositol-3,4,5-trisphosphate (PIP3) to phosphoinositol-4,5-bisphosphate (PIP2). We performed biochemical analyses of cellular phospholipids in endothelial cells to show that siRNA-mediated PTEN knockdown leads to a marked increase in PIP3. Because these complex lipids activate the small GTPase Rac1, we explored the role of PTEN in ADP-modulated Rac1 activation. We used a FRET biosensor for Rac1 to show that ADP-dependent Rac1 activation is blocked by siRNA-mediated PTEN knockdown. We then exploited a FRET biosensor for PIP3 to show that the striking ADP-dependent increase in intracellular PIP3 is entirely blocked by PTEN knockdown. These studies identify a key role for PTEN in the modulation of lipid mediators involved in ADP receptor-regulated endothelial signaling pathways involving eNOS activation in vascular endothelial cells. © 2013 Elsevier B.V.National Institutes of Health (HL46457, HL48743, GM36259); Ministerio de Economía y Competitividad; Fundación Renal “Iñigo Álvarez deToledo”; Fundación “Ramón Areces”Peer Reviewe

Therapeutic miR-506-3p Replacement in Pancreatic Carcinoma Leads to Multiple Effects including Autophagy, Apoptosis, Senescence, and Mitochondrial Alterations In Vitro and In Vivo

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality. Considering its very poor prognosis, novel treatment options are urgently needed. MicroRNAs (miRNAs) are involved in the regulation of various physiological and pathological processes. In tumors, aberrant downregulation of given miRNAs may result in pathological overexpression of oncogenes, rendering miRNA replacement as a promising therapeutic strategy. In different tumor entities, miRNA-506-3p (miR506-3p) has been ambivalently described as tumor suppressing or oncogenic. In PDAC, miR-506 is mainly considered as a tumor-suppressing miRNA. In this study, we extensively analyze the cellular and molecular effects of miRNA-506-3p replacement in different PDAC cell lines. Beyond profound antiproliferation and induction of cell death and autophagy, we describe new cellular miR506-3p effects, i.e., induction of senescence and reactive oxygen species (ROS), as well as alterations in mitochondrial potential and structure, and identify multiple underlying molecular effects. In a preclinical therapy study, PDAC xenograft-bearing mice were treated with nanoparticle-formulated miRNA-506 mimics. Profound tumor inhibition upon systemic miRNA-506 administration was associated with multiple cellular and molecular effects. This demonstrates miRNA replacement as a potential therapeutic option for PDAC patients. Due to its broad mechanisms of action on multiple relevant target genes, miR506-3p is identified as a particularly powerful tumor-inhibitory miRNA

Restoration of MARCK enhances chemosensitivity in cancer

Purpose!#!Increased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P!##!Methods!#!CRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P!##!Results!#!We found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU.!##!Conclusions!#!Cells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates