Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating triglyceride (TG) hydrolysis. The lack of ATGL results in TG accumulation in multiple tissues, underscoring the critical role of ATGL in maintaining lipid homeostasis. Recent evidence suggests that ATGL affects TG metabolism via activation of peroxisome proliferator-activated receptor α (PPARα). To investigate specific effects of intestinal ATGL on lipid metabolism we generated mice lacking ATGL exclusively in the intestine (ATGLiKO). We found decreased TG hydrolase activity and increased intracellular TG content in ATGLiKO small intestines. Intragastric administration of [3H]trioleate resulted in the accumulation of radioactive TG in the intestine, whereas absorption into the systemic circulation was unchanged. Intraperitoneally injected [3H]oleate also accumulated within TG in ATGLiKO intestines, indicating that ATGL mobilizes fatty acids from the systemic circulation absorbed by the basolateral side from the blood. Down-regulation of PPARα target genes suggested modulation of cholesterol absorption by intestinal ATGL. Accordingly, ATGL deficiency in the intestine resulted in delayed cholesterol absorption. Importantly, this study provides evidence that ATGL has no impact on intestinal TG absorption but hydrolyzes TGs taken up from the intestinal lumen and systemic circulation. Our data support the role of ATGL in modulating PPARα-dependent processes also in the small intestine.
The purpose of this study was to gain insights into the regulation of Epstein-Barr virus (EBV) gene transcription during the establishment of viral latency in B cells. During the early stages of EBV infection in B lymphocytes, transcription of six viral nuclear antigens (EBNAs) is initiated from an early promoter (Wp). This is followed by a switch of promoter usage to an upstream promoter, Cp, whose activity is autoregulated by both EBNA1 and EBNA2. Previously it was demonstrated that infection of primary B cells with EBNA2-negative (EBNA2-) EBNA4-mutant (EBNA4mut) virus resulted only in the expression of mutant EBNA4 protein and failure to express the other EBNA gene products (C. Rooney H. G. Howe, S. H. Speck, and G. Miller, J. Virol. 63:1531-1539, 1989). We extended this research to demonstrate that Wp-to-Cp switching did not occur upon infection of primary B cells with an EBNA2- EBNA4mut virus (M. Woisetschlaeger, X. W. Jin, C. N. Yandara, L. A. Furmanski, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991). Further characterization of this phenomenon led to the identification of an EBNA2-dependent enhancer upstream of Cp. On the basis of these data, a model was proposed in which initial transcription from Wp gives rise to the expression of EBNA2 and EBNA4, and then transcription is upregulated from Cp via the EBNA2- dependent enhancer (Woisetschlaeger et al., as noted above). Implicit in this model is that transcription of the EBNA1 and EBNA3a to -3c genes is dependent on the switch from Wp to Cp, since primary cells infected with EBNA2- EBNA4mut virus fail to switch and also fail to express these viral antigens. Here we critically evaluate this model and demonstrate, in contrast to the predictions of the model, that transcription of both the EBNA1 and EBNA2 genes precedes activation of Cp. Furthermore, the level of EBNA1 gene transcription was strongly reduced in primary B cells infected with EBNA2- EBNA4mut virus compared with that of cells infected with wild-type virus. Switching to Cp, as well as EBNA1 gene transcription, was observed upon infection of EBV-negative Burkitt's lymphoma (BL) cell lines with EBNA2- EBNA4mut virus, thus establishing a correlation between early EBNA1 gene transcription and upregulation of transcription initiation from Cp. However, in EBV-negative BL cell lines infected with EBNA2- EBNA4mut virus, transcription of the EBNA1 gene at early time points postinfection initiated from Qp, the EBNA1 gene promoter active in group I BL cells (B. C. Schaefer, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 92:10565-10569, 1995), rather than from Wp. The data support a model in which EBNA1 plays an important role in the cascade of events leading to successful switching from Wp to Cp and subsequent immortalization of the infected B cell.
The importance of peroxisomes for adipocyte function is poorly understood. Herein, we provide insights into the critical role of peroxin 16 (PEX16)-mediated peroxisome biogenesis in adipocyte development and lipid metabolism. Pex16 is highly expressed in adipose tissues and upregulated during adipogenesis of murine and human cells. We demonstrate that Pex16 is a target gene of the adipogenesis "master-regulator" PPARγ. Stable silencing of Pex16 in 3T3-L1 cells strongly reduced the number of peroxisomes while mitochondrial number was unaffected. Concomitantly, peroxisomal fatty acid (FA) oxidation was reduced, thereby causing accumulation of long- and very long-chain (polyunsaturated) FAs and reduction of odd-chain FAs. Further, Pex16-silencing decreased cellular oxygen consumption and increased FA release. Additionally, silencing of Pex16 impaired adipocyte differentiation, lipogenic and adipogenic marker gene expression, and cellular triglyceride stores. Addition of PPARγ agonist rosiglitazone and peroxisome-related lipid species to Pex16-silenced 3T3-L1 cells rescued adipogenesis. These data provide evidence that PEX16 is required for peroxisome biogenesis and highlights the relevance of peroxisomes for adipogenesis and adipocyte lipid metabolism.
The synthesis, structure, photophysics, and spectroscopic characterization of an organometallic rhenium multichromophore compound carrying a central 2,2′-bipyridyl acceptor moiety with additional phenylethynyl substituents conjugated at the 4,4′-positions of the acceptor ligands and its effect on the electron density of the central rhenium atom as metal center for CO2 reduction is reported. The results were compared to fac-(2,2′-bipyridyl)Re(CO)3Cl and fac-(5,5′-bisphenylethynyl-2,2′-bipyridyl)Re(CO)3Cl. Cyclovoltammetric studies and rotating disk electrochemistry were performed for electrochemical characterization. Ultraviolet and visible (UV-vis) absorption, Fourier transform infrared (FTIR), and luminescence measurements were carried out for a spectroscopic characterization and compared to theoretical calculations at the density functional theory (DFT) level. In addition, the rhenium complex fac-(4,4′-bisphenyl-ethynyl-2,2′-bipyridyl)-Re(CO)3Cl was used as a novel catalyst for the electrochemical reduction of CO2 in homogeneous solution. Results showed an 11-fold increase in the current density under CO2 saturation and a catalytic second-order rate constant for CO formation of about 560 M−1 s−1 on a Pt working electrode. For further characterization of the CO2 reduction capabilities, bulk controlled potential electrolysis experiments were performed using a CO2-saturated acetonitrile electrolyte solution. The headspace product gas analysis yields CO as main reduction product with faradaic efficiencies of about 12 % over 5-h electrolysis time.
Microglia, the immunocompetent cells of the CNS, rapidly respond to brain injury and disease by altering their morphology and phenotype to adopt an activated state. Microglia can exist broadly between two different states, namely the classical (M1) and the alternative (M2) phenotype. The first is characterized by the production of pro-inflammatory cytokines/chemokines and reactive oxygen and/or nitrogen species. In contrast, alternatively activated microglia are typified by an anti-inflammatory phenotype supporting wound healing and debris clearance. The objective of the present study was to determine the outcome of lysophosphatidic acid (LPA)-mediated signaling events on microglia polarization. LPA receptor expression and cyto-/chemokine mRNA levels in BV-2 and primary murine microglia (PMM) were determined by qPCR. M1/M2 marker expression was analyzed by Western blotting, immunofluorescence microscopy, or flow cytometry. Cyto-/chemokine secretion was quantitated by ELISA. BV-2 cells express LPA receptor 2 (LPA2), 3, 5, and 6, whereas PMM express LPA1, 2, 4, 5, and 6. We show that LPA treatment of BV-2 and PMM leads to a shift towards a pro-inflammatory M1-like phenotype. LPA treatment increased CD40 and CD86 (M1 markers) and reduced CD206 (M2 marker) expression. LPA increased inducible nitric oxide synthase (iNOS) and COX-2 levels (both M1), while the M2 marker Arginase-1 was suppressed in BV-2 cells. Immunofluorescence studies (iNOS, COX-2, Arginase-1, and RELMα) extended these findings to PMM. Upregulation of M1 markers in BV-2 and PMM was accompanied by increased cyto-/chemokine transcription and secretion (IL-1β, TNFα, IL-6, CCL5, and CXCL2). The pharmacological LPA5 antagonist TCLPA5 blunted most of these pro-inflammatory responses. LPA drives BV-2 and PMM towards a pro-inflammatory M1-like phenotype. Suppression by TCLPA5 indicates that the LPA/LPA5 signaling axis could represent a potential pharmacological target to interfere with microglia polarization in disease.
// Stefanie Schlager 1 , Carina Salomon 1 , Sabine Olt 1 , Christoph Albrecht 1 , Anja Ebert 2 , Oliver Bergner 1 , Johannes Wachter 1 , Francesca Trapani 1 , Daniel Gerlach 1 , Tilman Voss 1 , Anna Traunbauer 2 , Julian Jude 2 , Matthias Hinterndorfer 2 , Martina Minnich 2 , Norbert Schweifer 1 , Sophia M. Blake 1 , 3 , Vittoria Zinzalla 1 , Barbara Drobits 1 , Darryl B. McConnell 1 , Norbert Kraut 1 , Mark Pearson 1 , Johannes Zuber 2 , 4 and Manfred Koegl 1 1 Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria 2 Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Vienna, Austria 3 Current address: AstraZeneca AB, Gothenburg, Sweden 4 Medical University of Vienna, Vienna BioCenter (VBC), Vienna, Austria Correspondence to: Manfred Koegl, email: manfred.koegl@boehringer-ingelheim.com Keywords: BCL6; DLBCL; lymphoma; inducible CRISPR/Cas9; in vivo xenograft Received: May 24, 2019 Accepted: February 08, 2020 Published: March 03, 2020 ABSTRACT Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations. Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients. An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL. However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors. Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and in vivo . We show that BCL6 knock-out in SU-DHL-4 cells in vitro results in an anti-proliferative response 4–7 days after Cas9 induction that was characterized by cell cycle (G1) arrest. Conditional BCL6 deletion in established DLBCL tumors in vivo induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics. Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/Cas9 systems for probing oncogene addiction.
The Cover Feature illustrates the consequences of CO2 release into the atmosphere from man-made sources. With humanity re-emitting, within a couple of decades, large amounts of CO2 that was fixated in millions of years though natural photosynthesis, there is a clear need for cyclic usage of CO2 to create a sustainable future employing photo- and bio-electrocatalysis. More information can be found in the Review by D. H. Apaydin et al. on page 3094 in Issue 22, 2017 (DOI: 10.1002/cphc.201700148).
Abstract The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B-cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We have used structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also cause rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and antiproliferative effects than compounds that merely inhibited co-repressor interaction. The fact that the magnitude of effects elicited by this class of BCL6 degrading compounds greatly exceeds that of our equipotent non-degrading inhibitors offers exciting opportunities for the development of BCL6-based lymphoma therapeutics. To support further research, the most potent BCL6 degrading inhibitor is made freely available to the research community as an in vitro tool compound. Please see http://www.opnMe.com for further infos. Citation Format: Nina Kerres, Steffen Steurer, Stefanie Schlager, Gerd Bader, Maureen Caligiuri, Christian Dank, John R. Engen, Peter Ettmayer, Daniel Gerlach, Thomas Gerstberger, Bingsong Han, Roxana E. Iacob, Dirk Kessler, David R. Lancia, Mayer Moriz, Nikolai Mischerikow, Klaus Rumpel, Renate Schnitzer, Tilman Voss, Xiaozhang Zheng, Andreas Zoephel, Norbert Kraut, Darryl McConnell, Mark Pearson, Manfred Koegl. Chemically induced degradation of the transcription factor BCL6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3354.
