Inhibition of mitochondrial complex II by the anti-cancer agent lonidamine [Lipids]

October 31st, 2015 by

The anti-tumor agent Lonidamine (LND, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid) is known to interfere with energy yielding processes in cancer cells. However, the effect of LND on central energy metabolism has never been fully characterized. In this study, we report that significant amount of succinate is accumulated in LND treated cells. LND inhibits the formation of fumarate and malate and suppresses succinate-induced respiration of isolated mitochondria. Utilizing biochemical assays, we determined that LND inhibits the succinate-ubiquinone reductase (SQR) activity of respiratory complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species (ROS) through complex II, which reduced the viability of the DB-1 melanoma cell line. The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. Using stable isotope tracers in combination with isotopologue analysis, we showed LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate. Our findings on the previously uncharacterized effects of LND may provide potential combinational therapeutic approaches for targeting cancer metabolism.

Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (HYaluronan Binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts. [Metabolism]

October 30th, 2015 by

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We have recently reported that HYBID (HYaluronan Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, bFGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression, but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower-molecular-weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, bFGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high-molecular-weight HA by HAS up-regulation and HYBID down-regulation, and suggest that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients.
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Telomerase deficiency causes alveolar stem cell senescence-associated low-grade inflammation in lungs [Molecular Bases of Disease]

October 30th, 2015 by

Mutations of human telomerase RNA component (TERC) and telomerase reverse transcriptase (TERT) are associated with a subset of lung aging diseases, but the mechanisms by which TERC and TERT participate in lung diseases remain unclear. In this report, we show that knockout (KO) of the mouse gene TERC or TERT causes pulmonary alveolar stem cell replicative senescence, epithelial impairment, formation of alveolar sacs, and characteristic inflammatory phenotype. Deficiency in TERC or TERT causes a remarkable elevation in various proinflammatory cytokines, including IL-1, IL-6, CXCL15 (human IL-8 homolog), IL-10, TNF-α and monocyte chemotactic protein 1 (chemokine ligand 2, CCL2), decrease in TGF-β1 and TGFβRI receptor in the lungs, and spillover of IL-6 and CXCL15 into the bronchoalveolar lavage fluids. In addition to increased gene expressions of α-smooth muscle actin (α-SMA) and collagen 1α1 (Col1α1) suggesting myofibroblast differentiation, TERC deficiency also leads to marked cellular infiltrations of a mononuclear cell population positive for the leukocyte common antigen CD45 (LCA), low-affinity Fc receptor CD16/CD32, and pattern recognition receptor CD11b in the lungs. Our data demonstrate for the first time that telomerase deficiency triggers alveolar stem cell replicative senescence-associated low-grade inflammation, thereby driving pulmonary premature aging, alveolar sac formation and fibrotic lesion.

MiR-346 Up-regulates Argonaute 2 (AGO2) Protein Expression to Augment the Activity of Other MiRNAs and Contributes to Cervical Cancer Cell Malignancy [Cell Biology]

October 30th, 2015 by Guo, J., Lv, J., Liu, M., Tang, H.

microRNAs (miRNAs) are a class of post-transcriptional regulators of gene expression, and AGO2 is essential for miRNA activity. In this study, we focused on the regulation of AGO2 by miR-346 and the consequences in cervical cancer cells. miR-346 enhanced the expression of AGO2, resulting in the increased activity of other miRNAs and contributing to the malignancy of HeLa cells. GRSF1 participated in the regulation of AGO2 by miR-346, and the middle sequence of miR-346 was vital for synergy effect of miR-346 and GRSF1. We determined that miR-346 promoted the migration and invasion of HeLa cells. In summary, we are the first to report that AGO2 is positively regulated by miRNA and that GRSF1 participates in the miRNA pathway.
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Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate Receptor 2 in a Human Cholangiocarcinoma Cell Line [Signal Transduction]

October 30th, 2015 by

Cholangiocarcinoma (CCA) is a rare, but highly malignant primary hepatobiliary cancer with a very poor prognosis and limited treatment options. Our recent studies reported that conjugated bile acids (CBAs) promote the invasive growth of CCA via activation of sphingosine 1-phosphate receptor 2 (S1PR2). Cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is the most abundant prostaglandin in various human malignancies including CCA. Previous studies have indicated that COX-2 was highly expressed in CCA tissues, and the survival rate of CCA patients was negatively associated with high COX-2 expression levels. It has also been reported that CBAs induce COX-2 expression, while free bile acids inhibit COX-2 expression in CCA mouse models. However, the underlying cellular mechanisms and connection between S1PR2 and COX-2 expression in CCA cells have still not been fully elucidated. In the current study, we examined the role of S1PR2 in conjugated bile acid (taurocholate, TCA)-induced COX-2 expression in a human HuCCT1 CCA cell line and further identified the potential underlying cellular mechanisms. The results indicated that TCA-induced invasive growth of human CCA cells was correlated with S1PR2-medated upregulation of COX-2 expression and PGE2 production. Inhibition of S1PR2 activation with chemical antagonist (JTE-013) or downregulation of S1PR2 expression with gene-specific shRNA not only reduced COX-2 expression, but also inhibited TCA-induced activation of EGFR and ERK1/2/Akt-NF-kB signaling cascade. Conclusion: S1PR2 plays a critical role in TCA-induced COX-2 expression and CCA growth and may represent a novel therapeutic target for CCA.
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{alpha}-Defensins induce a post-translational modification of LDL that promotes atherosclerosis at normal levels of plasma cholesterol [Metabolism]

October 30th, 2015 by

Approximately one-half of patients who develop clinical atherosclerosis have normal or only modest elevations in plasma lipids, indicating additional mechanisms contribute to pathogenesis. In view of increasing evidence that inflammation contributes to atherogenesis, we studied the effect of human neutrophil α-defensins (α-defs) on low density lipoprotein (LDL) trafficking, metabolism, vascular deposition and atherogenesis using transgenic mice expressing human α-defs in their polymorphonuclear leukocytes (Def+/+). Def+/+ mice develop α-def/LDL complexes that accelerate the clearance of LDL from the circulation accompanied by enhanced vascular deposition and retention of LDL, induction of endothelial cathepsins, increased endothelial permeability to LDL and the development of lipid streaks in the aortic roots when fed a regular diet and at normal plasma levels of LDL. Transplantation of bone marrow from Def+/+ to WT mice increased LDL clearance, increased vascular permeability and increased vascular deposition of LDL, while transplantation of WT bone marrow to Def+/+ mice prevented these outcomes. The same outcome was obtained by treating Def+/+ mice with colchicine to inhibit release of α-defs. These studies identify a potential new link between inflammation and the development of atherosclerosis.
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Metabolic cooperation of glucose and glutamine is essential for the lytic cycle of obligate intracellular parasite Toxoplasma gondii [Metabolism]

October 30th, 2015 by Nitzsche, R., Zagoriy, V., Lucius, R., Gupta, N.

Toxoplasma gondii is a widespread protozoan parasite, infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here, we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the TCA cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. Growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine, but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells.
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Alternative Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and can Activate Cyclin-Dependent Kinase in Xenopus Oocytes [Molecular Bases of Disease]

October 30th, 2015 by

KV10.1 is a voltage-gated potassium channel expressed selectively in mammalian brain, but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lack the transmembrane domains of the channel, and would produce cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitate with the full-length channel and induce a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect is mechanistically different. E65 requires a tetramerization domain and induces a reduction in the overall expression of full-length KV10.1, while E70 mainly affects its glycosylation pattern. E65 triggers activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of non-canonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy.
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Kruppel homolog 1 inhibits insect metamorphosis via direct transcriptional repression of Broad-complex, a pupal specifier gene [Signal Transduction]

October 30th, 2015 by

The Broad-complex gene (BR-C) encodes transcription factors that dictate larval-pupal metamorphosis in insects. The expression of BR-C is induced by molting hormone (20-hydroxyecdysone, 20E), and this induction is repressed by juvenile hormone (JH), which exists during the premature larval stage. Kruppel homolog 1 gene (Kr-h1) has been known as a JH-early-inducible gene responsible for repression of metamorphosis; however, the functional relationship between Kr-h1 and repression of BR-C has remained unclear. To elucidate this relationship, we analyzed cis- and trans-elements involved in the repression of BR-C using a Bombyx mori cell line. In the cells, as observed in larvae, JH induced the expression of Kr-h1 and concurrently suppressed 20E-induced expression of BR-C. Forced expression of Kr-h1 repressed the 20E-dependent activation of the BR-C promoter in the absence of JH, and Kr-h1 RNAi inhibited the JH-mediated repression, suggesting that Kr-h1 controlled the repression of BR-C. A survey of the upstream sequence of BR-C gene revealed a Kr-h1 binding site (KBS) in the BR-C promoter. When KBS was deleted from the promoter, the repression of BR-C was abolished. Electrophoresis mobility shift demonstrated that two Kr-h1 molecules bound to KBS in the BR-C promoter. Based on these results, we conclude that Kr-h1 protein molecules directly bind to the KBS sequence in the BR-C promoter, and thereby repress 20E-dependent activation of the pupal specifier, BR-C. This study has revealed a considerable portion of the picture of JH signaling pathways from the reception of JH to the repression of metamorphosis.
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Non-equivalence of key positively charged residues of the free fatty acid 2 receptor in the recognition and function of agonist versus antagonist ligands [Protein Structure and Folding]

October 29th, 2015 by

Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of poorly digested carbohydrates. A key mediator of their actions is the G protein-coupled Free Fatty Acid 2 (FFA2) receptor and this has been suggested as a therapeutic target for the treatment of both metabolic and inflammatory diseases. However, a lack of understanding of the molecular determinants dictating how ligands bind to this receptor has hindered development. We have developed a novel radiolabelled FFA2 antagonist in order to probe ligand binding to FFA2 and in combination with mutagenesis and molecular modelling studies define how agonist and antagonist ligands interact with the receptor. Although both agonist and antagonist ligands contain negatively charged carboxylates that interact with two key positively charged arginine residues in transmembrane domains V and VII of FFA2, there are clear differences in how these interactions occur. Specifically, while agonists require interaction with both arginine residues to bind the receptor, antagonists require an interaction with only one of the two. Moreover, different chemical series of antagonist interact preferentially with different arginine residues. A homology model capable of rationalizing these observations was developed and provides a tool that will be invaluable for identifying improved FFA2 agonists and antagonists to further define function and therapeutic opportunities of this receptor.
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