BBA - Molecular Basis of Disease (v.1852, #7)

Intraneuronal accumulation of misfolded tau protein induces overexpression of Hsp27 in activated astrocytes by Peter Filipcik; Martin Cente; Norbert Zilka; Tomas Smolek; Jozef Hanes; Juraj Kucerak; Alena Opattova; Branislav Kovacech; Michal Novak (1219-1229).
Accumulation of misfolded forms of microtubule associated, neuronal protein tau causes neurofibrillary degeneration typical of Alzheimer's disease and other tauopathies. This process is accompanied by elevated cellular stress and concomitant deregulation of heat-shock proteins. We used a transgenic rat model of tauopathy to study involvement of heat shock protein 27 (Hsp27) in the process of neurofibrillary degeneration, its cell type specific expression and correlation with the amount of insoluble tau protein aggregates. The expression of Hsp27-mRNA is more than doubled and levels of Hsp27 protein tripled in aged transgenic animals with tau pathology. The data revealed a strong positive and highly significant correlation between Hsp27-mRNA and amount of sarkosyl insoluble tau. Interestingly, intracellular accumulation of insoluble misfolded tau protein in neurons was associated with overexpression of Hsp27 almost exclusively in reactive astrocytes, not in neurons. The topological dissociation of neuronally expressed pathological tau and the induction of astrocytic Hsp27, GFAP, and Vimentin along with up-regulation of microglia specific markers such as CD18, CD68 and C3 point to cooperation of astrocytes, microglia and neurons in response to intra-neuronal accumulation of insoluble tau. Our data suggest that over expression of Hsp27 represents a part of microglia-mediated astrocytic response mechanism in the process of neurofibrillary degeneration, which is not necessarily associated with neuroprotection and which in contrary may accelerate neurodegeneration in late stage of the disease. This phenomenon should be considered during development of disease modifying strategies for treatment of tauopathies and AD via regulation of activity of Hsp27.
Keywords: Truncated tau protein; Tauopathy; Alzheimer's disease; Hsp27; Transgenic rat; Astrocyte;

Blockage of ROS and NF-κB-mediated inflammation by a new chalcone L6H9 protects cardiomyocytes from hyperglycemia-induced injuries by Peng Zhong; Lianpin Wu; Yuanyuan Qian; Qilu Fang; Dandan Liang; Jingying Wang; Chunlai Zeng; Yi Wang; Guang Liang (1230-1241).
Increased oxidative stress and cardiac inflammation have been implicated in the pathogenesis of diabetic cardiomyopathy (DCM). We previously found that a novel chalcone derivative, L6H9, was able to reduce LPS-induced inflammatory response in macrophages. This study was designed to investigate its protective effects on DCM and the underlying mechanisms. H9C2 cells were cultured with DMEM containing 33 mmol/L of glucose in the presence or absence of L6H9. Pretreatment with L6H9 significantly reduced high glucose-induced inflammatory cytokine expression, ROS level increase, mitochondrial dysfunction, cell apoptosis, fibrosis, and hypertrophy in H9c2 cells, which may be mediated by NF-κB inhibition and Nrf2 activation. In mice with STZ-induced diabetes, oral administration of L6H9 at 20 mg/kg/day for 8 weeks significantly decreased the cardiac cytokine and ROS level, accompanied by decreasing cardiac apoptosis and hypertrophy, and, finally, improved histological abnormalities and fibrosis, without affecting the hyperglycemia. L6H9 also attenuated the diabetes-induced NF-κB activation and Nrf2 decrease in diabetic hearts. These results strongly suggest that L6H9 may have great therapeutic potential in the treatment of DCM via blockage of inflammation and oxidative stress. This study also provides a deeper understanding of the regulatory role of Nrf2 and NF-κB in DCM, indicating that they may be important therapeutic targets for diabetic complications.
Keywords: Diabetic cardiomyopathy; Chalcone derivative; Inflammation; Oxidative stress; NF-κB; Nrf2;

Role of fibroblast growth factor 21 in the early stage of NASH induced by methionine- and choline-deficient diet by Naoki Tanaka; Shogo Takahashi; Yuan Zhang; Kristopher W. Krausz; Philip B. Smith; Andrew D. Patterson; Frank J. Gonzalez (1242-1252).
Fibroblast growth factor 21 (FGF21) is a modulator of energy homeostasis and is increased in human nonalcoholic liver disease (NAFLD) and after feeding of methionine- and choline-deficient diet (MCD), a conventional inducer of murine nonalcoholic steatohepatitis (NASH). However, the significance of FGF21 induction in the occurrence of MCD-induced NASH remains undetermined. C57BL/6J Fgf21-null and wild-type mice were treated with MCD for 1 week. Hepatic Fgf21 mRNA was increased early after commencing MCD treatment independent of peroxisome proliferator-activated receptor (PPAR) α and farnesoid X receptor. While no significant differences in white adipose lipolysis were seen in both genotypes, hepatic triglyceride (TG) contents were increased in Fgf21-null mice, likely due to the up-regulation of genes encoding CD36 and phosphatidic acid phosphatase 2a/2c, involved in fatty acid (FA) uptake and diacylglycerol synthesis, respectively, and suppression of increased mRNAs encoding carnitine palmitoyl-CoA transferase 1α, PPARγ coactivator 1α, and adipose TG lipase, which are associated with lipid clearance in the liver. The MCD-treated Fgf21-null mice showed increased hepatic endoplasmic reticulum (ER) stress. Exposure of primary hepatocytes to palmitic acid elevated the mRNA levels encoding DNA damage-inducible transcript 3, an indicator of ER stress, and FGF21 in a PPARα-independent manner, suggesting that lipid-induced ER stress can enhance hepatic FGF21 expression. Collectively, FGF21 is elevated in the early stage of MCD-induced NASH likely to minimize hepatic lipid accumulation and ensuing ER stress. These results provide a possible mechanism on how FGF21 is increased in NAFLD/NASH.
Keywords: ER stress; Lipotoxicity; ATGL; PGC1α; PPARα;

Ethical issues with artificial nutrition of children with degenerative brain diseases by Alfried Kohlschütter; Carolina Riga; Dolores Crespo; José Manuel Torres; Victor Penchaszadeh; Angela Schulz (1253-1256).
This report highlights viewpoints of the authors and comments from the auditory at a workshop, held during the 14th international Congress on neuronal ceroid lipofuscinoses (NCL) in Córdoba, Argentina, on ethical aspects of artificial nutrition in children with degenerative brain diseases. The discussion centers on what constitutes the best interest of a patient whose personality was immature before the onset of the disease, who has become demented during its course and is unable to communicate his/her own positions and desires. There is wide consensus that in a child with advanced disease who cannot be fed naturally, decisions to withhold nutrition or to institute or stop artificial nutrition, should only be made by parents (or their representatives) who are adequately prepared on an intellectual and emotional level. We try to show that such decisions are highly individual but can be made in a rationally and emotionally acceptable way after a careful and prolonged dialogue between families and professionals. A checklist summarizes important considerations. This article is part of a Special Issue entitled: “Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease)”.
Keywords: Childhood dementia; Inborn error of metabolism; Neurodegeneration; Vital support;

Metformin and caloric restriction induce an AMPK-dependent restoration of mitochondrial dysfunction in fibroblasts from Fibromyalgia patients by Elísabet Alcocer-Gómez; Juan Garrido-Maraver; Pedro Bullón; Fabiola Marín-Aguilar; David Cotán; Angel M. Carrión; José Miguel Alvarez-Suarez; Francesca Giampieri; José Antonio Sánchez-Alcazar; Maurizio Battino; Mario D. Cordero (1257-1267).
Impaired AMPK is associated with a wide spectrum of clinical and pathological conditions, ranging from obesity, altered responses to exercise or metabolic syndrome, to inflammation, disturbed mitochondrial biogenesis and defective response to energy stress. Fibromyalgia (FM) is a world-wide diffused musculoskeletal chronic pain condition that affects up to 5% of the general population and comprises all the above mentioned pathophysiological states. Here, we tested the involvement of AMPK activation in fibroblasts derived from FM patients. AMPK was not phosphorylated in fibroblasts from FM patients and was associated with decreased mitochondrial biogenesis, reduced oxygen consumption, decreased antioxidant enzymes expression levels and mitochondrial dysfunction. However, mtDNA sequencing analysis did not show any important alterations which could justify the mitochondrial defects. AMPK activation in FM fibroblast was impaired in response to moderate oxidative stress. In contrast, AMPK activation by metformin or incubation with serum from caloric restricted mice improved the response to moderate oxidative stress and mitochondrial metabolism in FM fibroblasts. These results suggest that AMPK plays an essential role in FM pathophysiology and could represent the basis for a valuable new therapeutic target/strategy. Furthermore, both metformin and caloric restriction could be an interesting therapeutic approach in FM.
Keywords: Fibromyalgia; AMPK; Mitochondria; Oxidative stress; Metformin; Caloric restriction;

Role of Akt and Ca2 + on cell permeabilization via connexin43 hemichannels induced by metabolic inhibition by Daniela Salas; Carlos Puebla; Paul D. Lampe; Sergio Lavandero; Juan C. Sáez (1268-1277).
Connexin hemichannels are regulated under physiological and pathological conditions. Metabolic inhibition, a model of ischemia, promotes surface hemichannel activation associated, in part, with increased surface hemichannel levels, but little is known about its underlying mechanism. Here, we investigated the role of Akt on the connexin43 hemichannel's response induced by metabolic inhibition. In HeLa cells stably transfected with rat connexin43 fused to EGFP (HeLa43 cells), metabolic inhibition induced a transient Akt activation necessary to increase the amount of surface connexin43. The increase in levels of surface connexin43 was also found to depend on an intracellular Ca2+ signal increase that was partially mediated by Akt activation. However, the metabolic inhibition-induced Akt activation was not significantly affected by intracellular Ca2+ chelation. The Akt-dependent increase in connexin43 hemichannel activity in HeLa43 cells also occurred after oxygen-glucose deprivation, another ischemia-like condition, and in cultured cortical astrocytes (endogenous connexin43 expression system) under metabolic inhibition. Since opening of hemichannels has been shown to accelerate cell death, inhibition of Akt-dependent phosphorylation of connexin43 hemichannels could reduce cell death induced by ischemia/reperfusion.Display Omitted
Keywords: Surface hemichannels; Intracellular Ca2+; Oxygen-glucose deprivation; Akt activation;

Apelin protects against acute renal injury by inhibiting TGF-β1 by Hong Chen; Danyang Wan; Lin Wang; Anlin Peng; Hongdou Xiao; Robert B. Petersen; Chengyu Liu; Ling Zheng; Kun Huang (1278-1287).
Renal ischemia/reperfusion (I/R) injury is the most common cause of acute kidney injury, having a high rate of mortality and no effective therapy currently available. Apelin-13, a bioactive peptide, has been shown to inhibit the early lesions of diabetic nephropathy in several mouse models by us and others. To test whether apelin-13 protects against renal I/R induced injury, male rats were exposed to renal I/R injury with or without apelin-13 treatment for 3 days. Apelin-13 treatment markedly reduced the injury-induced tubular lesions, renal cell apoptosis, and normalized the injury induced renal dysfunction. Apelin-13 treatment inhibited the injury-induced elevation of inflammatory factors and Tgf-β1, as well as apoptosis. Apelin-13 treatment also inhibited the injury-induced elevation of histone methylation and Kmt2d, a histone methyltransferase of H3K4me2, following renal I/R injury. Furthermore, in cultured renal mesangial and tubular cells, apelin-13 suppressed the injury-induced elevation of Tgf-β1, apoptosis, H3K4me2 and Kmt2d under the in vitro hypoxia/reperfusion (H/R) conditions. Consistently, over-expression of apelin significantly inhibited H/R-induced elevation of TGF-β1, apoptosis, H3K4me2 and Kmt2d. The present study therefore suggests apelin-13 may be a therapeutic candidate for treating acute kidney injury.
Keywords: Apelin; Renal I/R injury; Histone methylation; TGF-β1;

Effect of azithromycin on the LPS-induced production and secretion of phospholipase A2 in lung cells by Eirini Kitsiouli; Georgia Antoniou; Helen Gotzou; Michalis Karagiannopoulos; Dimitris Basagiannis; Savvas Christoforidis; George Nakos; Marilena E. Lekka (1288-1297).
Azithromycin is a member of macrolides, utilized in the treatment of infections. Independently, these antibiotics also possess anti-inflammatory and immunomodulatory properties. Phospholipase A2 isotypes, which are implicated in the pathophysiology of inflammatory lung disorders, are produced by alveolar macrophages and other lung cells during inflammatory response and can promote lung injury by destructing lung surfactant. The aim of the study was to investigate whether in lung cells azithromycin can inhibit secretory and cytosolic phospholipases A2, (sPLA2) and (cPLA2), respectively, which are induced by an inflammatory trigger. In this respect, we studied the lipopolysaccharide (LPS)-mediated production or secretion of sPLA2 and cPLA2 from A549 cells, a cancer bronchial epithelial cell line, and alveolar macrophages, isolated from bronchoalveolar lavage fluid of ARDS and control patients without cardiopulmonary disease or sepsis. Pre-treatment of cells with azithromycin caused a dose-dependent decrease in the LPS-induced sPLA2-IIA levels in A549 cells. This inhibition was rather due to reduced PLA2G2A mRNA expression and secretion of sPLA2-IIA protein levels, as observed by western blotting and indirect immunofluorescence by confocal microscopy, respectively, than to the inhibition of the enzymic activity per se. On the contrary, azithromycin had no effect on the LPS-induced production or secretion of sPLA2-IIA from alveolar macrophages. The levels of LPS-induced c-PLA2 were not significantly affected by azithromycin in either cell type. We conclude that azithromycin exerts anti-inflammatory properties on lung epithelial cells through the inhibition of both the expression and secretion of LPS-induced sPLA2-IIA, while it does not affect alveolar macrophages.Display Omitted
Keywords: Azithromycin; Phospholipase A2; Lung epithelial cells; A549 cells; Alveolar macrophages; ARDS;

Protective effects of resveratrol on the inhibition of hippocampal neurogenesis induced by ethanol during early postnatal life by Le Xu; Yang Yang; Lixiong Gao; Jinghui Zhao; Yulong Cai; Jing Huang; Sheng Jing; Xiaohang Bao; Ying Wang; Junwei Gao; Haiwei Xu; Xiaotang Fan (1298-1310).
Ethanol (EtOH) exposure during early postnatal life triggers obvious neurotoxic effects on the developing hippocampus and results in long-term effects on hippocampal neurogenesis. Resveratrol (RSV) has been demonstrated to exert potential neuroprotective effects by promoting hippocampal neurogenesis. However, the effects of RSV on the EtOH-mediated impairment of hippocampal neurogenesis remain undetermined. Thus, mice were pretreated with RSV and were later exposed to EtOH to evaluate its protective effects on EtOH-mediated toxicity during hippocampal development. The results indicated that a brief exposure of EtOH on postnatal day 7 resulted in a significant impairment in hippocampal neurogenesis and a depletion of hippocampal neural precursor cells (NPCs). This effect was attenuated by pretreatment with RSV. Furthermore, EtOH exposure resulted in a reduction in spine density on the granular neurons of the dentate gyrus (DG), and the spines exhibited a less mature morphological phenotype characterized by a higher proportion of stubby spines and a lower proportion of mushroom spines. However, RSV treatment effectively reversed these responses. We further confirmed that RSV treatment reversed the EtOH-induced down-regulation of hippocampal pERK and Hes1 protein levels, which may be related to the proliferation and maintenance of NPCs. Furthermore, EtOH exposure in the C17.2 NPCs also diminished cell proliferation and activated apoptosis, which could be reversed by pretreatment of RSV. Overall, our results suggest that RSV pretreatment protects against EtOH-induced defects in neurogenesis in postnatal mice and may thus play a critical role in preventing EtOH-mediated toxicity in the developing hippocampus.
Keywords: Ethanol; Neurogenesis; Hippocampal development; Neural precursor cell; Resveratrol;

Sirtuins in vascular diseases: Emerging roles and therapeutic potential by Nunzia D'Onofrio; Milena Vitiello; Rosario Casale; Luigi Servillo; Alfonso Giovane; Maria Luisa Balestrieri (1311-1322).
Silent information regulator-2 (Sir-2) proteins, or sirtuins, are a highly conserved protein family of histone deacetylases that promote longevity by mediating many of the beneficial effects of calorie restriction which extends life span and reduces the incidence of cancer, cardiovascular disease (CVD), and diabetes. Here, we review the role of sirtuins (SIRT1-7) in vascular homeostasis and diseases by providing an update on the latest knowledge about their roles in endothelial damage and vascular repair mechanisms. Among all sirtuins, in the light of the numerous functions reported on SIRT1 in the vascular system, herein we discuss its roles not only in the control of endothelial cells (EC) functionality but also in other cell types beyond EC, including endothelial progenitor cells (EPC), smooth muscle cells (SMC), and immune cells. Furthermore, we also provide an update on the growing field of compounds under clinical evaluation for the modulation of SIRT1 which, at the state of the art, represents the most promising target for the development of novel drugs against CVD, especially when concomitant with type 2 diabetes.
Keywords: Sirtuins; Endothelium; Vascular dysfunction; Endothelial progenitor cell; Cardiovascular disease;

Interaction of thrombospondin1 and CD36 contributes to obesity-associated podocytopathy by Wenpeng Cui; Hasiyeti Maimaitiyiming; Qi Zhou; Heather Norman; Changcheng Zhou; Shuxia Wang (1323-1333).
Obesity is associated with podocyte injury and the development of proteinuria. Elevated plasma free fatty acid is one of the characteristics of obesity and has been linked to podocyte dysfunction. However, the mechanisms remain unclear. In the current study, we examined the effect of saturated free fatty acid (FFA) on human podocyte apoptosis and function in vitro. The mechanism and its in vivo relevance were also determined. We found that FFA treatment induced human podocyte apoptosis and dysfunction, which was associated with increased expression of a matricellular protein-thrombospondin1 (TSP1). FFA stimulated TSP1 expression in podocytes at the transcriptional levels through activation of MAPK pathway. Addition of purified TSP1 to cell culture media induced podocyte apoptosis and dysfunction. Tis effect is though a TGF-β independent mechanism. Moreover, peptide treatment to block TSP1 binding to its receptor-CD36 attenuated FFA induced podocyte apoptosis, suggesting that TSP1/CD36 interaction mediates FFA-induced podocyte apoptosis. Importantly, using a diet-induced obese mouse model, in vivo data demonstrated that obesity-associated podocyte apoptosis and dysfunction were attenuated in TSP1 deficient mice as well as in CD36 deficient mice. Taken together, these studies provide novel evidence that the interaction of TSP1 with its receptor CD36 contributes to obesity — associated podocytopathy.Display Omitted
Keywords: Podocyte apoptosis; Free fatty acid; CD36; Obesity; TSP1;

The flavo-oxidase QSOX1 supports vascular smooth muscle cell migration and proliferation: Evidence for a role in neointima growth by Beatriz E. Borges; Márcia H. Appel; Axel R. Cofré; Maiara L. Prado; Chelin A. Steclan; Frédéric Esnard; Silvio M. Zanata; Francisco R.M. Laurindo; Lia S. Nakao (1334-1346).
Quiescin sulfhydryl oxidase 1 (QSOX1) is a flavoenzyme largely present in the extracellular milieu whose physiological functions and substrates are not known. QSOX1 has been implicated in the regulation of tumor cell survival, proliferation and migration, in addition to extracellular matrix (ECM) remodeling. However, data regarding other pathophysiological conditions are still lacking. Arterial injury by balloon catheter is an established model of post-angioplasty restenosis. This technique induces neointima formation due to migration and proliferation of vascular smooth muscle cells (VSMC), followed by ECM synthesis and remodeling. Here, we show that QSOX1 knockdown inhibited VSMC migration and proliferation in vitro. In contrast, QSOX1 overexpression stimulated these processes. While migration could be induced by the incubation of cells with the active recombinant QSOX1, proliferation was induced by addition of the active and also of an inactive mutant QSOX1 protein. The proliferation induced by both recombinants was independent of intracellular hydrogen peroxide and dependent of the MEK/ERK pathway. To recapitulate in vivo VSMC pathophysiology, balloon-induced arterial injury was performed. The expression of QSOX1 in the neointimal layer of balloon-injured rat carotids was high and peaked at 14 days post-injury. In vivo QSOX1 knockdown led to a significant decrease in PCNA expression at day 14 post-injury and a decreased intima/media area ratio at day 21 post-injury, compared with scrambled siRNA transfection. In summary, our findings demonstrate that QSOX1 induces VSMC migration and proliferation in vitro and contributes to neointima thickening in balloon-injured rat carotids.
Keywords: Quiescin sulfhydryl oxidase; Cell proliferation; Cell migration; Vascular smooth muscle cell; Neointima;

Iron is essential for several vital biological processes. Its deficiency or overload drives to the development of several pathologies. To maintain iron homeostasis, the organism controls the dietary iron absorption by enterocytes, its recycling by macrophages and storage in hepatocytes. These processes are mainly controlled by hepcidin, a liver-derived hormone which synthesis is regulated by iron levels, inflammation, infection, anemia and erythropoiesis. Besides the systemic regulation of iron metabolism mediated by hepcidin, cellular regulatory processes also occur. Cells are able to regulate themselves the expression of the iron metabolism-related genes through different post-transcriptional mechanisms, such as the alternative splicing, microRNAs, the IRP/IRE system and the proteolytic cleavage. Whenever those mechanisms are disturbed, due to genetic or environmental factors, iron homeostasis is disrupted and iron related pathologies may arise.
Keywords: Iron; Hepcidin; Post-transcriptional regulation; Hemochromatosis; Iron deficiency;

Transient receptor potential channel M2 contributes to neointimal hyperplasia in vascular walls by Xiaochen Ru; Changbo Zheng; Qiannan Zhao; Hui-Yao Lan; Yu Huang; Song Wan; Yasuo Mori; Xiaoqiang Yao (1360-1371).
A hallmark of atherosclerosis is progressive intimal thickening (namely neointimal hyperplasia), which leads to occlusive vascular diseases. Over-production of reactive oxygen species (ROS) and alteration of Ca2 + signaling are among the key factors contributing to neointimal growth. In the present study, we investigated the role of TRPM2, a ROS-sensitive Ca2 + entry channel, in neointimal hyperplasia.Perivascular cuffs were used to induce neointimal hyperplasia in rat/mouse arteries. Immunostaining showed numerous TRPM2-positive smooth muscle cells in neointimal regions. ROS were over-produced and PCNA-positive proliferating cells were numerous in the neointimal regions. The neointimal hyperplasia was substantially reduced in Trpm2 knockout mice compared with wild-type mice. In the cultured rat/mouse aortic smooth muscle cells, H2O2 treatment was found to stimulate cell proliferation and migration. The effect of H2O2 was reduced by a TRPM2-specific blocking antibody TM2E3 or Trpm2 knockout. The signaling molecules downstream of TRPM2 were found to be Axl and Akt.These data suggest a critical functional role of TRPM2 in the progression of neointimal hyperplasia. The study also highlights the possibility of targeting TRPM2 as a potential therapeutic option for the treatment of occlusive vascular diseases.
Keywords: Neointimal hyperplasia; TRPM2; Vascular smooth muscle cell; Migration; Proliferation;

A novel mutation in PNLIP causes pancreatic triglyceride lipase deficiency through protein misfolding by András Szabó; Xunjun Xiao; Margaret Haughney; Alyssa Spector; Miklós Sahin-Tóth; Mark E. Lowe (1372-1379).
Congenital pancreatic triglyceride lipase (PNLIP) deficiency is a rare disorder with uncertain genetic background as most cases were described before gene sequencing was readily available. Recently, two brothers with PNLIP deficiency were found to carry a homozygous missense mutation, c.662C > T (p.T221M) in the PNLIP gene (J. Lipid Res. 2014. 55:307–312). Molecular modeling suggested the substitution would change the orientation of residues in the catalytic site and disrupt the function of p.T221M PNLIP. To test the effect of the p.T221M mutation on PNLIP function, we expressed wild-type and p.T221M PNLIP in human embryonic kidney (HEK) 293A cells and dexamethasone-differentiated AR42J rat acinar cells. In both cellular models, wild-type PNLIP was secreted into the conditioned medium where it was readily detectable by protein staining, immunoblot or lipase activity assays. In contrast, mutant p.T221M was not secreted into the medium, but it was present in cell lysates where it accumulated in the insoluble fraction. Intracellular retention of mutant p.T221M resulted in endoplasmic reticulum (ER) stress as measured by elevated XBP1 splicing and increased levels of ER chaperones. Our results demonstrate that the presence of methionine at position 221 in the PNLIP protein sequence causes misfolding and aggregation of the p.T221M mutant inside the cell. The consequent loss of enzyme secretion adequately explains the clinical phenotype of PNLIP deficiency reported for homozygous carriers of p.T221M. Furthermore, the ability of mutant p.T221M to induce ER stress suggests that this form of PNLIP deficiency might cause acinar cell damage as well.
Keywords: Lipase; Fat digestion; Protein misfolding; Endoplasmic reticulum stress response;

Acetylcholinesterase is associated with a decrease in cell proliferation of hepatocellular carcinoma cells by Benjamín Pérez-Aguilar; Cecilio J. Vidal; Guillermina Palomec; Fernando García-Dolores; María Concepción Gutiérrez-Ruiz; Leticia Bucio; José Luis Gómez-Olivares; Luis Enrique Gómez-Quiroz (1380-1387).
Acetylcholinesterase (AChE), the enzyme that rapidly splits acetylcholine into acetate and choline, presents non-cholinergic functions through which may participate in the control of cell proliferation and apoptosis. These two features are relevant in cancer, particularly in hepatocellular carcinoma (HCC), a very aggressive liver tumor with high incidence and poor prognosis in advanced stages. Here we explored the relation between acetylcholinesterase and HCC growth by testing the influence of AChE on proliferation of Huh-7 and HepG2 cell lines, addressed in monolayer cultures, spheroid formation and human liver tumor samples. Results showed a clear relation in AChE expression and cell cycle progression, an effect which depended on cell confluence. Inhibition of AChE activity led to an increase in cell proliferation, which was associated with downregulation of p27 and cyclins. The fact that Huh-7 and HepG2 cell lines provided similar results lent weight to the relationship of AChE expression with cell cycle progression in hepatoma cell lines at least. Human liver tumor samples exhibited a decrease in AChE activity as compared with normal tissue. The evidence presented herein provides additional support for the proposed tumor suppressor role of AChE, which makes it a potential therapeutic target in therapies against hepatocellular carcinoma.Display Omitted
Keywords: Acetylcholinesterase; Cell cycle; HCC; HepG2 cell; Huh-7 cell; Liver;

Involvement of activated SUMO-2 conjugation in cardiomyopathy by Eun Young Kim; Yi Zhang; Bo Ye; Ana Maria Segura; Ilimbek Beketaev; Yutao Xi; Wei Yu; Jiang Chang; Faqian Li; Jun Wang (1388-1399).
Sumoylation is a posttranslational modification that regulates a wide spectrum of cellular activities. Cardiomyopathy is the leading cause of heart failure. Whether sumoylation, particularly SUMO-2/3 conjugation, is involved in cardiomyopathy has not been investigated. We report here that SUMO-2/3 conjugation was elevated in the human failing hearts, and we investigated the impact of increased SUMO-2 conjugation on heart function by using the gain-of-function approach in mice, in which cardiac specific expression of constitutively active SUMO-2 was governed by alpha myosin heavy chain promoter (MHC-SUMO-2 transgenic, SUMO-2-Tg). Four of five independent SUMO-2-Tg mouse lines exhibited cardiomyopathy with various severities, ranging from acute heart failure leading to early death to the development of chronic cardiomyopathy with aging. We further revealed that SUMO-2 directly regulated apoptotic process by at least partially targeting calpain 2 and its natural inhibitor calpastatin. SUMO conjugation to calpain 2 promoted its enzymatic activity, and SUMO attachment to calpastatin mainly promoted its turnover and altered its subcellular distribution. Thus, enhanced SUMO-2 conjugation led to increased apoptosis and played a pathogenic role in the development of cardiomyopathy and heart failure.
Keywords: SUMO; Cardiomyopathy; Apoptosis; Calpain 2; Calpastatin;

On the mechanism underlying ethanol-induced mitochondrial dynamic disruption and autophagy response by Luis Bonet-Ponce; Sara Saez-Atienzar; Carmen da Casa; Miguel Flores-Bellver; Jorge M. Barcia; Javier Sancho-Pelluz; Francisco J. Romero; Joaquín Jordan; María F. Galindo (1400-1409).
We have explored the mechanisms underlying ethanol-induced mitochondrial dynamics disruption and mitophagy. Ethanol increases mitochondrial fission in a concentration-dependent manner through Drp1 mitochondrial translocation and OPA1 proteolytic cleavage. ARPE-19 (a human retinal pigment epithelial cell line) cells challenged with ethanol showed mitochondrial potential disruptions mediated by alterations in mitochondrial complex IV protein level and increases in mitochondrial reactive oxygen species production. In addition, ethanol activated the canonical autophagic pathway, as denoted by autophagosome formation and autophagy regulator elements including Beclin1, ATG5-ATG12 and P-S6 kinase. Likewise, autophagy inhibition dramatically increased mitochondrial fission and cell death, whereas autophagy stimulation rendered the opposite results, placing autophagy as a cytoprotective response aimed to remove damaged mitochondria. Interestingly, although ethanol induced mitochondrial Bax translocation, this episode was associated to cell death rather than mitochondrial fission or autophagy responses. Thus, Bax required 600 mM ethanol to migrate to mitochondria, a concentration that resulted in cell death. Furthermore, mouse embryonic fibroblasts lacking this protein respond to ethanol by undergoing mitochondrial fission and autophagy but not cytotoxicity. Finally, by using the specific mitochondrial-targeted scavenger MitoQ, we revealed mitochondria as the main source of reactive oxygen species that trigger autophagy activation. These findings suggest that cells respond to ethanol activating mitochondrial fission machinery by Drp1 and OPA1 rather than bax, in a manner that stimulates cytoprotective autophagy through mitochondrial ROS.
Keywords: Apoptosis; Alcohol; Oxidative stress; Drp-1; Bax;

Altered ROS production, NF-κB activation and interleukin-6 gene expression induced by electrical stimulation in dystrophic mdx skeletal muscle cells by Carlos Henríquez-Olguín; Francisco Altamirano; Denisse Valladares; José R. López; Paul D. Allen; Enrique Jaimovich (1410-1419).
Duchenne muscular dystrophy is a fatal X-linked genetic disease, caused by mutations in the dystrophin gene, which cause functional loss of this protein. This pathology is associated with an increased production of reactive oxygen (ROS) and nitrogen species. The aim of this work was to study the alterations in NF-κB activation and interleukin-6 (IL-6) expression induced by membrane depolarization in dystrophic mdx myotubes. Membrane depolarization elicited by electrical stimulation increased p65 phosphorylation, NF-κB transcriptional activity and NF-κB-dependent IL-6 expression in wt myotubes, whereas in mdx myotubes it had the opposite effect. We have previously shown that depolarization-induced intracellular Ca2 + increases and ROS production are necessary for NF-κB activation and stimulation of gene expression in wt myotubes. Dystrophic myotubes showed a reduced amplitude and area under the curve of the Ca2 + transient elicited by electrical stimulation. On the other hand, electrical stimuli induced higher ROS production in mdx than wt myotubes, which were blocked by NOX2 inhibitors. Moreover, mRNA expression and protein levels of the NADPH oxidase subunits: p47phox and gp91phox were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50 μM H2O2 increased NF-κB activity; after administration of 100 and 200 μM H2O2 there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H2O2, with a significant effect of 100 μM and 200 μM, suggesting that ROS levels are critical for NF-κB activity. Prior blockage with NOX2 inhibitors blunted the effects of electrical stimuli in both NF-κB activation and IL-6 expression. Finally, to ascertain whether stimulation of NF-κB and IL-6 gene expression by the inflammatory pathway is also impaired in mdx myotubes, we studied the effect of lipopolysaccharide on both NF-κB activation and IL-6 expression. Exposure to lipopolysaccharide induced a dramatic increase in both NF-κB activation and IL-6 expression in both wt and mdx myotubes, suggesting that the altered IL-6 gene expression after electrical stimulation in mdx muscle cells is due to dysregulation of Ca2 + release and ROS production, both of which impinge on NF-κB signaling. IL-6 is a key metabolic modulator that is released by the skeletal muscle to coordinate a multi-systemic response (liver, muscle, and adipocytes) during physical exercise; the alteration of this response in dystrophic muscles may contribute to an abnormal response to contraction and exercise.
Keywords: Duchenne muscular dystrophy; NF-κB; Reactive oxygen species; Interleukin-6; Membrane depolarization; Calcium;

The regulation of mitochondrial biogenesis is under the control of nuclear genes including the master Mitochondrial Transcription Factor A (TFAM). Recent evidence suggests that the expression of TFAM is regulated by microRNAs (miRNAs) in various cellular contexts. Here, we show that hsa-miR-155-5p, a prominent miRNA encoded in chromosome 21, controls the expression of TFAM at the post-transcriptional level. In human fibroblasts derived from a diploid donor, downregulation of TFAM by hsa-miR-155-5p decreased mitochondrial DNA (mtDNA) content. In contrast, downregulation of TFAM by hsa-miR-155-5p did not decrease mtDNA content in fibroblasts derived from a donor with Down syndrome (DS, trisomy 21). In line, downregulation of mitochondrial TFAM levels through hsa-miR-155-5p decreased mitochondrial mass in diploid fibroblasts but not in trisomic cells. Due to the prevalence of mitochondrial dysfunction and cardiac abnormalities in subjects with DS, we examined the presence of potential associations between hsa-miR-155-5p and TFAM expression in heart samples from donors with and without DS. There were significant negative associations between hsa-miR-155-5p and TFAM expression in heart samples from donors with and without DS. These results suggest that regulation of TFAM by hsa-miR-155-5p impacts mitochondrial biogenesis in the diploid setting but not in the DS setting.
Keywords: TFAM; Hsa-miR-155-5p; Mitochondrial biogenesis; Down syndrome;

Oxidative stress involving changes in Nrf2 and ER stress in early stages of Alzheimer's disease by Sandra I. Mota; Rui O. Costa; Ildete L. Ferreira; Isabel Santana; Gladys L. Caldeira; Carmela Padovano; Ana C. Fonseca; Inês Baldeiras; Catarina Cunha; Liliana Letra; Catarina R. Oliveira; Cláudia M.F. Pereira; Ana Cristina Rego (1428-1441).
Oxidative stress and endoplasmic reticulum (ER) stress have been associated with Alzheimer's disease (AD) progression. In this study we analyzed whether oxidative stress involving changes in Nrf2 and ER stress may constitute early events in AD pathogenesis by using human peripheral blood cells and an AD transgenic mouse model at different disease stages. Increased oxidative stress and increased phosphorylated Nrf2 (p(Ser40)Nrf2) were observed in human peripheral blood mononuclear cells (PBMCs) isolated from individuals with mild cognitive impairment (MCI). Moreover, we observed impaired ER Ca2+ homeostasis and increased ER stress markers in PBMCs from MCI individuals and mild AD patients. Evidence of early oxidative stress defense mechanisms in AD was substantiated by increased p(Ser40)Nrf2 in 3 month-old 3xTg-AD male mice PBMCs, and also with increased nuclear Nrf2 levels in brain cortex. However, SOD1 protein levels were decreased in human MCI PBMCs and in 3xTg-AD mice brain cortex; the latter further correlated with reduced SOD1 mRNA levels. Increased ER stress was also detected in the brain cortex of young female and old male 3xTg-AD mice. We demonstrate oxidative stress and early Nrf2 activation in AD human and mouse models, which fails to regulate some of its targets, leading to repressed expression of antioxidant defenses (e.g., SOD-1), and extending to ER stress. Results suggest markers of prodromal AD linked to oxidative stress associated with Nrf2 activation and ER stress that may be followed in human peripheral blood mononuclear cells.
Keywords: Alzheimer's disease; Peripheral blood mononuclear cell; Lymphocyte; Mild cognitive impairment; Oxidative stress; Calcium homeostasis;

Medium-chain triglycerides (MCT) are widely applied in the treatment of long-chain fatty acid oxidation disorders. Previously it was shown that long-term MCT supplementation strongly affects lipid metabolism in mice. We here investigate sex-specific effects in mice with very-long-chain-acyl-CoA dehydrogenase (VLCAD) deficiency in response to a long-term MCT modified diet. We quantified blood lipids, acylcarnitines, glucose, insulin and free fatty acids, as well as tissue triglycerides in the liver and skeletal muscle under a control and an MCT diet over 1 year. In addition, visceral and hepatic fat content and muscular intramyocellular lipids (IMCL) were assessed by in vivo 1H magnetic resonance spectroscopy (MRS) techniques. The long-term application of an MCT diet induced a marked alteration of glucose homeostasis. However, only VLCAD−/− female mice developed a severe metabolic syndrome characterized by marked insulin resistance, dyslipidemia, severe hepatic and visceral steatosis, whereas VLCAD−/− males seemed to be protected and only presented with milder insulin resistance. Moreover, the highly saturated MCT diet is associated with a decreased hepatic stearoyl-CoA desaturase 1 (SCD1) activity in females aggravating the harmful effects of a saturated MCT diet. Long-term MCT supplementation deeply affects lipid metabolism in a sexual dimorphic manner resulting in a severe metabolic syndrome only in female mice. These findings are striking since the first signs of insulin resistance already occur in female VLCAD−/− mice during their reproductive period. How these metabolic adaptations are finally regulated needs to be determined. More important, the relevance of these findings for humans under these dietary modifications needs to be investigated.
Keywords: VLCAD-deficiency; MCT supplementation; Sexual dimorphism; Metabolic syndrome;

Opposing roles of miR-21 and miR-29 in the progression of fibrosis in Duchenne muscular dystrophy by Simona Zanotti; Sara Gibertini; Maurizio Curcio; Paolo Savadori; Barbara Pasanisi; Lucia Morandi; Ferdinando Cornelio; Renato Mantegazza; Marina Mora (1451-1464).
Excessive extracellular matrix deposition progressively replacing muscle fibres is the endpoint of most severe muscle diseases. Recent data indicate major involvement of microRNAs in regulating pro- and anti-fibrotic genes. To investigate the roles of miR-21 and miR-29 in muscle fibrosis in Duchenne muscle dystrophy, we evaluated their expression in muscle biopsies from 14 patients, and in muscle-derived fibroblasts and myoblasts. In Duchenne muscle biopsies, miR-21 expression was significantly increased, and correlated directly with COL1A1 and COL6A1 transcript levels. MiR-21 expression was also significantly increased in Duchenne fibroblasts, more so after TGF-β1 treatment. In Duchenne fibroblasts the expression of miR-21 target transcripts PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SPRY-1 (Sprouty homolog 1) was significantly reduced; while collagen I and VI transcript levels and soluble collagen production were significantly increased. MiR-29a and miR-29c were significantly reduced in Duchenne muscle and myoblasts, and miR-29 target transcripts, COL3A1, FBN1 and YY1, significantly increased. MiR-21 silencing in mdx mice reduced fibrosis in the diaphragm muscle and in both Duchenne fibroblasts and mdx mice restored PTEN and SPRY-1 expression, and significantly reduced collagen I and VI expression; while miR-29 mimicking in Duchenne myoblasts significantly decreased miR-29 target transcripts. These findings indicate that miR-21 and miR-29 play opposing roles in Duchenne muscle fibrosis and suggest that pharmacological modulation of their expression has therapeutic potential for reducing fibrosis in this condition.
Keywords: MiR-21; Fibroblast; MiR-29; Myoblast; Fibrosis; Duchenne muscular dystrophy; mdx mouse;

Inhibition of PMCA activity by tau as a function of aging and Alzheimer's neuropathology by María Berrocal; Isaac Corbacho; María Vázquez-Hernández; Jesús Ávila; M. Rosario Sepúlveda; Ana M. Mata (1465-1476).
Ca2+-ATPases are plasma membrane and intracellular membrane transporters that use the energy of ATP hydrolysis to pump cytosolic Ca2+ out of the cell (PMCA) or into internal stores. These pumps are the main high-affinity Ca2+ systems involved in the maintenance of intracellular free Ca2+ at the properly low level in eukaryotic cells. The failure of neurons to keep optimal intracellular Ca2+ concentrations is a common feature of neurodegeneration by aging and aging-linked neuropathologies, such as Alzheimer's disease (AD). This disease is characterized by the accumulation of β-amyloid senile plaques and neurofibrillary tangles of tau, a protein that plays a key role in axonal transport. Here we show a novel inhibition of PMCA activity by tau which is concentration-dependent. The extent of inhibition significantly decreases with aging in mice and control human brain membranes, but inhibition profiles were similar in AD-affected brain membrane preparations, independently of age. No significant changes in PMCA expression and localization with aging or neuropathology were found. These results point out a link between Ca2+-transporters, aging and neurodegeneration mediated by tau protein.
Keywords: Tau; Calcium; PMCA; Aging; Alzheimer´s disease;

miR-155-dependent regulation of mammalian sterile 20-like kinase 2 (MST2) coordinates inflammation, oxidative stress and proliferation in vascular smooth muscle cells by Zhan Yang; Bin Zheng; Yu Zhang; Ming He; Xin-hua Zhang; Dong Ma; Ruo-nan Zhang; Xiao-li Wu; Jin-kun Wen (1477-1489).
In response to vascular injury, inflammation, oxidative stress, and cell proliferation often occur simultaneously in vascular tissues. We previously observed that microRNA-155 (miR-155), which is implicated in proliferation and inflammation is involved in neointimal hyperplasia; however, the molecular mechanisms by which it regulates these processes remain largely unknown. In this study, we observed that vascular smooth muscle cell (VSMC) proliferation and neointimal formation in wire-injured femoral arteries were reduced by the loss of miR-155 and increased by the gain of miR-155. The proliferative effect of miR-155 was also observed in cultured VSMCs. Notably, expression of the miR-155-target protein mammalian sterile 20-like kinase 2 (MST2) was increased in the injured arteries of miR-155−/− mice. miR-155 directly repressed MST2 and thus activated the extracellular signal-regulated kinase (ERK) pathway by promoting an interaction between RAF proto-oncogene serine/threonine-protein kinase (Raf-1) and mitogen-activated protein kinase kinase (MEK) and stimulating inflammatory and oxidative stress responses; together, these effects lead to VSMC proliferation and vascular remodeling. Our data reveal that MST2 mediates miR-155-promoted inflammatory and oxidative stress responses by altering the interaction of MEK with Raf-1 and MST2 in response to vascular injury. Therefore, suppression of endogenous miR-155 might be a novel therapeutic strategy for vascular injury and remodeling.Display Omitted
Keywords: VSMC; Proliferation; miR-155; MST2; Inflammation; Oxidative stress;

Celf1 regulates cell cycle and is partially responsible for defective myoblast differentiation in myotonic dystrophy RNA toxicity by Xiaoping Peng; Xiaopeng Shen; Xuanying Chen; Rui Liang; Alon R. Azares; Yu Liu (1490-1497).
Myotonic dystrophy is a neuromuscular disease of RNA toxicity. The disease gene DMPK harbors expanded CTG trinucleotide repeats on its 3′-UTR. The transcripts of this mutant DMPK led to misregulation of RNA-binding proteins including MBNL1 and Celf1. In myoblasts, CUG-expansion impaired terminal differentiation. In this study, we formally tested how the abundance of Celf1 regulates normal myocyte differentiation, and how Celf1 expression level mediates CUG-expansion RNA toxicity-triggered impairment of myocyte differentiation. As the results, overexpression of Celf1 largely recapitulated the defects of myocytes with CUG-expansion, by increasing myocyte cycling. Knockdown of endogenous Celf1 level led to precocious myotube formation, supporting a negative connection between Celf1 abundance and myocyte terminal differentiation. Finally, knockdown of Celf1 in myocyte with CUG-expansion led to partial rescue, by promoting cell cycle exit. Our results suggest that Celf1 plays a distinctive and negative role in terminal myocyte differentiation, which partially contribute to DM1 RNA toxicity. Targeting Celf1 may be a valid strategy in correcting DM1 muscle phenotypes, especially for congenital cases.
Keywords: Celf1; CUGBP1; Myotonic dystrophy; Differentiation; Myoblast;

Atherosclerosis, the underlying cause of myocardial infarction and thrombotic cerebrovascular events, is responsible for the majority of deaths in westernized societies. Mortality from this disease is also increasing at a marked rate in developing countries due to the acquisition of a westernized lifestyle accompanied with elevated rates of obesity and diabetes. Atherosclerosis is recognized as a chronic inflammatory disorder associated with lipid accumulation and the development of fibrotic plaques within the walls of medium and large arteries. A range of immune cells, such as macrophages and T-lymphocytes, through the action of various cytokines, such as interleukins-1 and -33, transforming growth factor-β and interferon-γ, orchestrates the inflammatory response in this disease. The disease is also characterized by marked dysfunction in lipid homeostasis and signaling pathways that control the inflammatory response. This review will discuss the molecular basis of atherosclerosis with particular emphasis on the roles of the immune cells and cytokines along with the dysfunctional lipid homeostasis and cell signaling associated with this disease.
Keywords: Atherosclerosis; Inflammation; Lipoproteins; Cytokines; Signaling; Immune cells;

Fenofibrate insulates diacylglycerol in lipid droplet/ER and preserves insulin signaling transduction in the liver of high fat fed mice by Stanley M.H. Chan; Xiao-Yi Zeng; Ruo-Qiong Sun; Eunjung Jo; Xiu Zhou; Hao Wang; Songpei Li; Aimin Xu; Matthew J. Watt; Ji-Ming Ye (1511-1519).
Hepatic steatosis is often associated with insulin resistance as a hallmark of the metabolic syndrome in the liver. The present study investigated the effects of PPARα activation induced by fenofibrate (FB) on the relationship of insulin resistance and hepatic steatosis in mice fed a high-fat (HF) diet, which increases lipid influx into the liver. Mice were fed HF diet to induce insulin resistance and hepatic steatosis with or without FB. FB activated PPARα and ameliorated HF diet-induced glucose intolerance and hepatic insulin resistance without altering either hepatic steatosis or inflammation signaling (JNK or IKK). Interestingly, FB treatment simultaneously increased fatty acid (FA) synthesis (50%) and oxidation (66%, both p  < 0.01) into intermediate lipid metabolites, suggesting a FA oxidation-synthesis cycling in operation. Associated with these effects, diacylglycerols (DAGs) were sequestered within the lipid droplet/ER compartment, thus reducing their deposition in the cellular membrane, which is known to impair insulin signal transduction. These findings suggest that the reduction in membrane DAGs (rather than total hepatic steatosis) may be critical for the protection by fenofibrate-induced PPARα activation against hepatic insulin resistance induced by dietary fat.A. High fat feeding increases FA supply cause hepatic steatosis and insulin resistance.B. Activation of PPARα by fenofibrate repartition diacylglycerols from the plasma membrane into the lipid droplet/ER to relieve hepatic insulin resistance in the presence of hepatosteatosis.Display Omitted
Keywords: PPARα; FA oxidation; FA synthesis; DAG repartitioning; Insulin signaling;

p-SMAD2/3 and DICER promote pre-miR-21 processing during pressure overload-associated myocardial remodeling by Raquel García; J. Francisco Nistal; David Merino; Nathan L. Price; Carlos Fernández-Hernando; Javier Beaumont; Arantxa González; María A. Hurlé; Ana V. Villar (1520-1530).
Transforming growth factor-β (TGF-β) induces miR-21 expression which contributes to fibrotic events in the left ventricle (LV) under pressure overload. SMAD effectors of TGF-β signaling interact with DROSHA to promote primary miR-21 processing into precursor miR-21 (pre-miR-21). We hypothesize that p-SMAD-2 and -3 also interact with DICER1 to regulate the processing of pre-miR-21 to mature miR-21 in cardiac fibroblasts under experimental and clinical pressure overload. The subjects of the study were mice undergoing transverse aortic constriction (TAC) and patients with aortic stenosis (AS). In vitro, NIH-3T3 fibroblasts transfected with pre-miR-21 responded to TGF-β1 stimulation by overexpressing miR-21. Overexpression and silencing of SMAD2/3 resulted in higher and lower production of mature miR-21, respectively. DICER1 co-precipitated along with SMAD2/3 and both proteins were up-regulated in the LV from TAC-mice. Pre-miR-21 was isolated bound to the DICER1 maturation complex. Immunofluorescence analysis revealed co-localization of p-SMAD2/3 and DICER1 in NIH-3T3 and mouse cardiac fibroblasts. DICER1-p-SMAD2/3 protein–protein interaction was confirmed by in situ proximity ligation assay. Myocardial up-regulation of DICER1 constituted a response to pressure overload in TAC-mice. DICER mRNA levels correlated directly with those of TGF-β1, SMAD2 and SMAD3. In the LV from AS patients, DICER mRNA was up-regulated and its transcript levels correlated directly with TGF-β1, SMAD2, and SMAD3. Our results support that p-SMAD2/3 interacts with DICER1 to promote pre-miR-21 processing to mature miR-21. This new TGFβ-dependent regulatory mechanism is involved in miR-21 overexpression in cultured fibroblasts, and in the pressure overloaded LV of mice and human patients.Display Omitted
Keywords: Pre-miR-21; p-SMAD2/3; Myocardial fibrosis; Pressure overload; DICER; TGF-β;

Redefining the roles of mitochondrial DNA-encoded subunits in respiratory Complex I assembly by Rasika Vartak; Janice Deng; Hezhi Fang; Yidong Bai (1531-1539).
Respiratory Complex I deficiency is implicated in numerous degenerative and metabolic diseases. In particular, mutations in several mitochondrial DNA (mtDNA)-encoded Complex I subunits including ND4, ND5 and ND6 have been identified in several neurological diseases. We previously demonstrated that these subunits played essential roles in Complex I assembly which in turn affected mitochondrial function. Here, we carried out a comprehensive study of the Complex I assembly pathway. We identified a new Complex I intermediate containing both membrane and matrix arms at an early assembly stage. We find that lack of the ND6 subunit does not hinder membrane arm formation; instead it recruits ND1 and ND5 enters the intermediate. While ND4 is important for the formation of the newly identified intermediate, the addition of ND5 stabilizes the complex and is required for the critical transition from Complex I to supercomplex assembly. As a result, the Complex I assembly pathway has been redefined in this study.
Keywords: Mitochondrial DNA mutations; Respiratory Complex I; Assembly; ND4; ND5; ND6;

EGFR over-expression in non-small cell lung cancers harboring EGFR mutations is associated with marked down-regulation of CD82 by Chi-Hwa Yang; Hsiao-Chin Chou; Yu-Ning Fu; Chi-Ling Yeh; Hui-Wen Cheng; Il-Chi Chang; Ko-Jiunn Liu; Gee-Chen Chang; Ting-Fen Tsai; Shih-Feng Tsai; Hui-Ping Liu; Yi-Cheng Wu; Ya-Ting Chen; Shiu-Feng Huang; Yi-Rong Chen (1540-1549).
Epidermal growth factor receptor (EGFR) gene mutations are strongly associated with lung adenocarcinoma and favorable response to EGFR tyrosine kinase inhibitor. The mutated EGFR proteins (EGFRs) are hyper-phosphorylated and refractory to receptor down-regulation. To address the discrepancy between hyper-phosphorylation and lack of down-regulation of mutant EGFRs, we have examined the expression of EGFR negative regulators in non-small cell lung cancer (NSCLC) cell lines. We found that NSCLC cell lines expressing mutant EGFRs often had low expression of various negative regulators for EGFR. Among them, tumor suppressor CD82 was up-regulated by wild type (WT) EGFR but down-regulated by mutant EGFRs. Reconstitution of CD82 exerted stronger suppressive effects on mutant EGFRs than on WT EGFR. Active exportation of CD82 through the exosome was one of the mechanisms involved in achieving the overall CD82 down-regulation in mutant EGFR-expressing lung cancer cell lines. Over-expression of mutant EGFR protein frequently occurred in the lung cancer tissues of mutant EGFR-transgenic mice and also associated with CD82 down-regulation. Immunoblot analyses on the tumor tissues from 23 lung adenocarcinoma patients (12 with WT EGFR, and 11 with mutant EGFRs) also identified significantly stronger down-regulation of CD82 in tumors with mutant EGFRs than WT. Our data indicate that CD82 down-regulation could be a critical step involved in the EGFR over-expression and the stronger tumorigenic activity triggered by EGFR mutations. Up-regulation of the CD82 level may become a promising new treatment strategy for lung adenocarcinoma.
Keywords: EGFR mutation; CD82; Lung cancer; Exosome;

Carbon monoxide protects against hepatic ischemia/reperfusion injury by modulating the miR-34a/SIRT1 pathway by Hyo Jeong Kim; Yeonsoo Joe; Jae Kyoung Yu; Yingqing Chen; Sun Oh Jeong; Nithya Mani; Gyeong Jae Cho; Hyun-Ock Pae; Stefan W. Ryter; Hun Taeg Chung (1550-1559).
Hepatic ischemia/reperfusion (I/R) injury can arise as a complication of liver surgery and transplantation. Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, modulates inflammation and apoptosis in response to oxidative stress. SIRT1, which is regulated by p53 and microRNA-34a (miR-34a), can modulate non-alcoholic fatty liver disease, fibrosis and cirrhosis. Since carbon monoxide (CO) inhalation can protect against hepatic I/R, we hypothesized that CO could ameliorate hepatic I/R injury by regulating the miR-34a/SIRT1 pathway. Livers from mice pretreated with CO, or PFT, a p53 inhibitor, displayed reduced production of pro-inflammatory mediators, including TNF-α, iNOS, interleukin (IL)-6, and IL-1β after hepatic I/R injury. SIRT1 expression was increased by CO or PFT in the liver after I/R, whereas acetylated p65, p53 levels, and miR-34a expression were decreased. CO increased SIRT1 expression by inhibiting miR-34a. Both CO and PFT diminished pro-inflammatory cytokines production in vitro. Knockdown of SIRT1 in LPS-stimulated macrophages increased NF-κB acetylation, and increased pro-inflammatory cytokines. CO treatment reduced miR-34a expression and increased SIRT1 expression in oxidant-challenged hepatocytes; and rescued SIRT1 expression in p53-expressing or miR-34a transfected cells. In response to CO, enhanced SIRT1 expression mediated by miR-34a inhibition protects against liver damage through p65/p53 deacetylation, which may mediate inflammatory responses and hepatocellular apoptosis. The miR-34a/SIRT1 pathway may represent a therapeutic target for hepatic injury.
Keywords: Carbon monoxide; p53; miR-34a; SIRT1; Liver;