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Oberoi-Khanuja T.K.,Goethe University Frankfurt | Karreman C.,University of Konstanz | Larisch S.,Haifa University | Rapp U.R.,Max Planck Institute For Herz Und Lungenforschung | Rajalingam K.,Goethe University Frankfurt
Journal of Biological Chemistry | Year: 2012

Inhibitor of apoptosis (IAPs) proteins are characterized by the presence of evolutionarily conserved baculoviral inhibitor of apoptosis repeat (BIR) domains, predominantly known for their role in inhibiting caspases and, thereby, apoptosis. We have shown previously that multi-BIR domain-containing IAPs, cellular IAPs, and X-linked IAP can control tumor cell migration by directly regulating the protein stability of C-RAF kinase. Here, we extend our observations to a single BIR domain containing IAP family member melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-RAF and that ML-IAP depletion leads to an increase in C-RAF protein levels, MAPK activation, and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-RAF stability and cell migration. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Source

Mollmann H.,Kerckhoff Heart Center | Nef H.M.,Kerckhoff Heart Center | Kostin S.,Max Planck Institute For Herz Und Lungenforschung | Dragu A.,Albert Ludwigs University of Freiburg | And 9 more authors.
International Journal of Cardiology | Year: 2010

Background: It is unknown whether the increased B-type natriuretic peptide (BNP) values found in ischemic heart disease are triggered directly by ischemia or whether they are caused indirectly by ischemia through diastolic contractures or regional wall motion abnormalities. Therefore, we investigated the BNP expression in isolated human muscle strips under conditions of ischemia with and without mechanical stress. Methods: Muscle strips (n = 90) were isolated from human right atria (n = 46). Contractures were induced by oxygen and glucose withdrawal. In 18 muscle strips contractures were prevented by means of butanedione monoxime (BDM). Sarcomere lengths were measured by electron microscopy (n = 12). The gene expression and protein amount of BNP were determined and compared to control muscle strips contracting under physiological conditions. Results: Hypoxia significantly decreased systolic force and induced diastolic contractures. This mechanical stress could be prevented in the group treated with BDM as evidenced by electron microscopy. Ischemia significantly increased BNP expression in both groups as evidenced by Northern blot analysis and immunohistochemistry. This increase was independent from mechanical stress. Conclusion: Our results indicate that ischemia is a potent mechanism for the expression of BNP. The increase in BNP expression under ischemic conditions is independent from concomitant mechanical alterations. © 2009 Elsevier Ireland Ltd. Source

Schafer A.,German Cancer Research Center | Schomacher L.,German Cancer Research Center | Barreto G.,German Cancer Research Center | Barreto G.,Max Planck Institute For Herz Und Lungenforschung | And 3 more authors.
PLoS ONE | Year: 2010

Gemcitabine is a cytotoxic cytidine analog, which is widely used in anti-cancer therapy. One mechanism by which gemcitabine acts is by inhibiting nucleotide excision repair (NER). Recently NER was implicated in Gadd45 mediated DNA demethylation and epigenetic gene activation. Here we analyzed the effect of gemcitabine on DNA demethylation. We find that gemcitabine inhibits specifically Gadd45a mediated reporter gene activation and DNA demethylation, similar to the topoisomerase I inhibitor camptothecin, which also inhibits NER. In contrast, base excision repair inhibitors had no effect on DNA demethylation. In Xenopus oocytes, gemcitabine inhibits DNA repair synthesis accompanying demethylation of oct4. n mammalian cells, gemcitabine induces DNA hypermethylation and silencing of MLH1. The results indicate that gemcitabine induces epigenetic gene silencing by inhibiting repair mediated DNA demethylation. Thus, gemcitabine can function epigenetically and provides a tool to manipulate DNA methylation. © 2010 Schäfer et al. Source

Kohlstedt K.,Goethe University Frankfurt | Trouvain C.,Goethe University Frankfurt | Boettger T.,Max Planck Institute For Herz Und Lungenforschung | Shi L.,Goethe University Frankfurt | And 2 more authors.
Circulation Research | Year: 2013

RATIONALE: High-angiotensin-converting enzyme (ACE)-levels are associated with cardiovascular disease, but little is known about the regulation of its expression. OBJECTIVE: To assess the molecular mechanisms regulating endothelial ACE expression focusing on the role of the AMP-activated protein kinase (AMPK) and miR-143/145. METHODS AND RESULTS: Shear stress decreased ACE expression in cultured endothelial cells, an effect prevented by downregulating AMPKα2 but not AMPKα1. AMPKα2-/- mice expressed higher ACE levels than wild-type littermates resulting in impaired hindlimb vasodilatation to the ACE substrate, bradykinin. The latter response was also evident in animals lacking the AMPKα2 subunit only in endothelial cells. In cultured endothelial cells, miR-143/145 levels were increased by shear stress in an AMPKα2-dependent manner, and miR-143/145 overexpression decreased ACE expression. The effect of shear stress was unrelated to an increase in miR-143/145 promoter activity and transcription but could be attributed to post-transcriptional regulation of precursor-miR-143/145 by AMPKα2. The AMPK substrate, p53, can enhance the post-transcriptional processing of several microRNAs, including miR-143/145. We found that shear stress elicited the AMPKα2-dependent phosphorylation of p53 (on Ser15), and that p53 downregulation prevented the shear stress-induced decrease in ACE expression. Streptozotocin-induced diabetes mellitus in mice was studied as a pathophysiological model of altered AMPK activity. Diabetes mellitus increased tissue phosphorylation of the AMPK substrates, p53 and acetyl-coenzyme A carboxylase, changes that correlated with increased miR-143/145 levels and decreased ACE expression. CONCLUSIONS: AMPKα2 suppresses endothelial ACE expression via the phosphorylation of p53 and upregulation of miR-143/145. Post-transcriptional regulation of miR-143/145 may contribute to the vascular complications associated with diabetes mellitus. Copyright © 2013 American Heart Association, Inc. Source

Weissmann N.,Justus Liebig University | Sydykov A.,Justus Liebig University | Kalwa H.,University of Marburg | Kalwa H.,Harvard University | And 20 more authors.
Nature Communications | Year: 2012

Lung ischaemia-reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2y/-) or the classical transient receptor potential channel 6 (TRPC6-/-) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca 2+ influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2 y/- cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE. © 2012 Macmillan Publishers Limited. All rights reserved. Source

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