Showing papers in "The FASEB Journal in 2013"
TL;DR: In this paper, the authors discuss the importance of chromatin structure and organization in eukaryotic genomes and their importance in maintaining the integrity of both genome and epigenome integrity with severe consequences for the organism.
Abstract: Stability and function of eukaryotic genomes are closely linked to chromatin structure and organization. During cell division the entire genome must be accurately replicated and the chromatin landscape reproduced on new DNA. Chromatin and nuclear structure influence where and when DNA replication initiates, whereas the replication process itself disrupts chromatin and challenges established patterns of genome regulation. Specialized replication-coupled mechanisms assemble new DNA into chromatin, but epigenome maintenance is a continuous process taking place throughout the cell cycle. If DNA synthesis is perturbed, cells can suffer loss of both genome and epigenome integrity with severe consequences for the organism.
509 citations
TL;DR: Diet‐induced paternal obesity modulates sperm microRNA content and germ cell methylation status, which are potential signals that program offspring health and initiate the transmission of obesity and impaired metabolic health to future generations.
Abstract: Obesity is highly prevalent, and its incidence is increasing. The previous study showing a major effect of paternal obesity on metabolic health of offspring is confounded by comorbidity with diabetes. Therefore, we investigated the effect of diet-induced paternal obesity, in the absence of diabetes, on the metabolic health of two resultant generations and the molecular profiles of the testes and sperm. Founder (F0) male C57BL6 mice were fed either a high-fat diet (HFD) or a control diet (CD); n = 10/diet for a period of 10 wk. Testis expression of mRNA/microRNAs was analyzed by microarray and qPCR and sperm microRNA abundance by qPCR. Two subsequent generations were generated by mating F0 and then F1 mice to CD mice, and their metabolic health was investigated. All mice, other than F0 males, were maintained on a CD. HFD feeding induced paternal obesity with a 21% increase in adiposity, but not overt diabetes, and initiated intergenerational transmission of obesity and insulin resistance in two generations of offspring. This distinct phenotypic constellation is either partially or fully transmitted to both female and male F1 offspring and further transmitted through both parental lineages to the F2 generation, with a heightened effect on female F1 offspring (+67% in adiposity) and their F2 sons (+24% in adiposity). Founder male obesity altered the testes expression of 414 mRNAs by microarray and 11 microRNAs by qPCR, concomitant with alterations in sperm microRNA content and a 25% reduction in global methylation of germ cell DNA. Diet-induced paternal obesity modulates sperm microRNA content and germ cell methylation status, which are potential signals that program offspring health and initiate the transmission of obesity and impaired metabolic health to future generations. This study implicates paternal obesity in the transgenerational amplification of obesity and type 2 diabetes in humans.
477 citations
TL;DR: An overview of the main characteristics and approaches used to identify, isolate, and characterize cancer stem cells from solid tumors is provided.
Abstract: Primary tumors are responsible for 10% of cancer deaths In most cases, the main cause of mortality is the formation of metastases Accumulating evidence suggests that a subpopulation of tumor cells with distinct stem-like properties is responsible for tumor initiation, invasive growth, and metastasis formation This population is defined as cancer stem cells (CSCs) Existing therapies have enhanced the length of survival after diagnosis of cancer but have completely failed in terms of recovery CSCs appear to be resistant to chemotherapy, may remain quiescent for extended periods, and have affinity for hypoxic environments The CSCs can be identified and isolated by different methodologies, including isolation by CSC-specific cell surface marker expression, detection of side population phenotype by Hoechst 33342 exclusion, assessment of their ability to grow as floating spheres, and aldehyde dehydrogenase (ALDH) activity assay None of the methods mentioned are exclusively used to isolate the solid tumor CSCs, highlighting the imperative to delineate more specific markers or to use combinatorial markers and methodologies This review provides an overview of the main characteristics and approaches used to identify, isolate, and characterize CSCs from solid tumors
350 citations
TL;DR: It is demonstrated that increased basal autophagy is required for endurance exercise training‐induced skeletal muscle adaptation and improvement of physical performance and revealed that endurance exerciseTraining‐induced increases in basal autophile, including mitophagy, only take place if an enhanced oxidative phenotype is achieved.
Abstract: Pathological and physiological stimuli, including acute exercise, activate autophagy; however, it is unknown whether exercise training alters basal levels of autophagy and whether autophagy is required for skeletal muscle adaptation to training. We observed greater autophagy flux (i.e., a combination of increased LC3-II/LC3-I ratio and LC3-II levels and reduced p62 protein content indicating a higher rate of initiation and resolution of autophagic events), autophagy protein expression (i.e., Atg6/Beclin1, Atg7, and Atg8/LC3) and mitophagy protein Bnip3 expression in tonic, oxidative muscle compared to muscles of either mixed fiber types or of predominant glycolytic fibers in mice. Long-term voluntary running (4 wk) resulted in increased basal autophagy flux and expression of autophagy proteins and Bnip3 in parallel to mitochondrial biogenesis in plantaris muscle with mixed fiber types. Conversely, exercise training promoted autophagy protein expression with no significant increases of autophagy flux and m...
349 citations
TL;DR: It is suggested that the mean size of focal adhesions robustly and precisely predicts cell speed independently of focalAdhesion surface density and molecular composition.
Abstract: Focal adhesions are large protein complexes organized at the basal surface of cells, which physically connect the extracellular matrix to the cytoskeleton and have long been speculated to mediate cell migration. However, whether clustering of these molecular components into focal adhesions is actually required for these proteins to regulate cell motility is unclear. Here we use quantitative microscopy to characterize descriptors of focal adhesion and cell motility for mouse embryonic fibroblasts and human fibrosarcoma cells, across a wide range of matrix compliance and following genetic manipulations of focal adhesion proteins (vinculin, talin, zyxin, FAK, and paxilin). This analysis reveals a tight, biphasic gaussian relationship between mean size of focal adhesions (not their number, surface density, or shape) and cell speed. The predictive power of this relationship is comprehensively validated by disrupting nonfocal adhesion proteins (α-actinin, F-actin, and myosin II) and subcellular organelles (mitochondria, nuclear DNA, etc.) not known to affect either focal adhesions or cell migration. This study suggests that the mean size of focal adhesions robustly and precisely predicts cell speed independently of focal adhesion surface density and molecular composition.
293 citations
TL;DR: The study evaluated longitudinal changes in an individual's microbiome by sampling 279 participants twice and 100 individuals 3 times, yielding 11,174 primary specimens, from which 12,479 DNA samples were submitted to 4 centers for metagenomic sequencing.
Abstract: The Human Microbiome Project used rigorous good clinical practice standards to complete comprehensive body site sampling in healthy 18- to 40-yr-old adults, creating an unparalleled reference set of microbiome specimens. To ensure that specimens represented minimally perturbed microbiomes, we first screened potential participants using exclusion criteria based on health history, including the presence of systemic diseases (e.g., hypertension, cancer, or immunodeficiency or autoimmune disorders), use of potential immunomodulators, and recent use of antibiotics or probiotics. Subsequent physical examinations excluded individuals based on body mass index (BMI), cutaneous lesions, and oral health. We screened 554 individuals to enroll 300 (149 men and 151 women, mean age 26 yr, mean BMI 24 kg/m2, 20.0% racial minority, and 10.7% Hispanic). We obtained specimens from the oral cavity, nares, skin, gastrointestinal tract, and vagina (15 specimens from men and 18 from women). The study evaluated longitudinal changes in an individual's microbiome by sampling 279 participants twice (mean 212 d after the first sampling; range 30-359 d) and 100 individuals 3 times (mean 72 d after the second sampling; range 30-224 d). This sampling strategy yielded 11,174 primary specimens, from which 12,479 DNA samples were submitted to 4 centers for metagenomic sequencing. Our clinical design and well-defined reference cohort has laid a foundation for microbiome research.—Aagaard, K., Petrosino, J., Keitel, W., Watson, M., Katancik, J., Garcia, N., Patel, S., Cutting, M., Madden, T., Hamilton, H., Harris, E., Gevers, D., Simone, G., McInnes, P., Versalovic, J. The Human Microbiome Project strategy for comprehensive sampling of the human microbiome and why it matters.
290 citations
TL;DR: It is shown that 2 hr of severe heat stress triggers global pausing of translation elongation at around codon 65 on most mRNAs in both mouse and human cells, and suggests that regulation oftranslation elongation in general, and by chaperones in particular, represents a major component of cellular stress responses.
Abstract: Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation. Heat shocked cells globally inhibit p...
277 citations
TL;DR: A paradigm of muscle‐fat crosstalk mediated by Fndc5, which is up‐regulated and secreted from muscle to induce beige cell markers and the browning of WAT in Mstn–/– mice is defined.
Abstract: Myostatin (Mstn) is predominantly expressed in skeletal muscles and plays important roles in regulating muscle growth and development, as well as fat deposition. Mstn-knockout (Mstn(-/-)) mice exhibit increased muscle mass due to both hypertrophy and hyperplasia, and leaner body composition due to reduced fat mass. Here, we show that white adipose tissue (WAT) of Mstn(-/-) develops characteristics of brown adipose tissue (BAT) with dramatically increased expression of BAT signature genes, including Ucp1 and Pgc1α, and beige adipocyte markers Tmem26 and CD137. Strikingly, the observed browning phenotype is non-cell autonomous and is instead driven by the newly defined myokine irisin (Fndc5) secreted from Mstn(-/-) skeletal muscle. Within the muscle, Mstn(-/-) leads to increased expression of AMPK and its phosphorylation, which subsequently activates PGC1α and Fndc5. Together, our study defines a paradigm of muscle-fat crosstalk mediated by Fndc5, which is up-regulated and secreted from muscle to induce beige cell markers and the browning of WAT in Mstn(-/-) mice. These results suggest that targeting muscle Mstn and its downstream signaling represents a therapeutic approach to treat obesity and type 2 diabetes.
267 citations
TL;DR: The tendon concentrations of 14C approximately reflected the atmospheric levels present during the first 17 yr of life, indicating that the tendon core is formed during height growth and is essentially not renewed thereafter, compared with 14C levels in muscle, which indicated continuous turnover.
Abstract: Tendons are often injured and heal poorly. Whether this is caused by a slow tissue turnover is unknown, since existing data provide diverging estimates of tendon protein half-life that range from 2 mo to 200 yr. With the purpose of determining life-long turnover of human tendon tissue, we used the 14C bomb-pulse method. This method takes advantage of the dramatic increase in atmospheric levels of 14C, produced by nuclear bomb tests in 1955–1963, which is reflected in all living organisms. Levels of 14C were measured in 28 forensic samples of Achilles tendon core and 4 skeletal muscle samples (donor birth years 1945–1983) with accelerator mass spectrometry (AMS) and compared to known atmospheric levels to estimate tissue turnover. We found that Achilles tendon tissue retained levels of 14C corresponding to atmospheric levels several decades before tissue sampling, demonstrating a very limited tissue turnover. The tendon concentrations of 14C approximately reflected the atmospheric levels present during the first 17 yr of life, indicating that the tendon core is formed during height growth and is essentially not renewed thereafter. In contrast, 14C levels in muscle indicated continuous turnover. Our observation provides a fundamental premise for understanding tendon function and pathology, and likely explains the poor regenerative capacity of tendon tissue.—Heinemeier, K. M., Schjerling, P., Heinemeier, J., Magnusson, S. P., Kjaer, M. Lack of tissue renewal in human adult Achilles tendon is revealed by nuclear bomb 14C.
258 citations
TL;DR: It is shown that polysulfides induce Ca2+ influx by activating transient receptor potential (TRP)A1 channels in rat astrocytes and that the maximum response was induced at 0.5 μM, which is 1/320 of the concentration of H2S required to achieve a response of similar magnitude.
Abstract: Accumulating evidence shows that hydrogen sulfide (H2S) has a variety of physiological functions. H2S is produced from cysteine by 3 sulfurtransferases. H2S, in turn, generates polysulfides, the functions of which are not well understood. H2S induces Ca(2+) influx in astrocytes, a type of glia. However, the receptor that mediates the response has not been identified. Here, we have shown that polysulfides induce Ca(2+) influx by activating transient receptor potential (TRP)A1 channels in rat astrocytes (EC50 91 nM, Hill coefficient value 1.77±0.26) and that the maximum response was induced at 0.5 μM, which is 1/320 of the concentration of H2S required to achieve a response of similar magnitude (160 μM, EC50 116 μM). TRPA1-selective agonists, allyl isothiocyanate and cinnamaldehyde, induced Ca(2+) influx, and responses to polysulfides were suppressed by TRPA1-selective inhibitors, HC-030031 and AP-18, as well as by siRNAs selective to TRPA1. The present study suggests that polysulfides are possible H2S-derived signaling molecules that stimulate TRP channels in the brain.
256 citations
TL;DR: It is suggested that astrocyte activation limits plaque growth and attenuates plaque‐related dystrophic neurites and accelerates plaque pathogenesis in APP/PS1 mice.
Abstract: The accumulation of aggregated amyloid-β (Aβ) in amyloid plaques is a neuropathological hallmark of Alzheimer's disease (AD). Reactive astrocytes are intimately associated with amyloid plaques; however, their role in AD pathogenesis is unclear. We deleted the genes encoding two intermediate filament proteins required for astrocyte activation-glial fibrillary acid protein (Gfap) and vimentin (Vim)-in transgenic mice expressing mutant human amyloid precursor protein and presenilin-1 (APP/PS1). The gene deletions increased amyloid plaque load: APP/PS1 Gfap(-/-)Vim(-/-) mice had twice the plaque load of APP/PS1 Gfap(+/+)Vim(+/+) mice at 8 and 12 mo of age. APP expression and soluble and interstitial fluid Aβ levels were unchanged, suggesting that the deletions had no effect on APP processing or Aβ generation. Astrocyte morphology was markedly altered by the deletions: wild-type astrocytes had hypertrophied processes that surrounded and infiltrated plaques, whereas Gfap(-/-)Vim(-/-) astrocytes had little process hypertrophy and lacked contact with adjacent plaques. Moreover, Gfap and Vim gene deletion resulted in a marked increase in dystrophic neurites (2- to 3-fold higher than APP/PS1 Gfap(+/+)Vim(+/+) mice), even after normalization for amyloid load. These results suggest that astrocyte activation limits plaque growth and attenuates plaque-related dystrophic neurites. These activities may require intimate contact between astrocyte and plaque.
TL;DR: It is concluded that an endogenous intramitochondrial H2S‐producing pathway, governed by 3‐MST, complements and balances the bioenergetic role of Krebs cycle‐derived electron donors.
Abstract: It is well established that exposure of mammalian cells to hydrogen sulfide (H(2)S) suppresses mitochondrial function by inhibiting cytochrome-c oxidase (CcOX; complex IV). However, recent experimental data show that administration of H(2)S to mammalian cells can serve as an electron donor and inorganic source of energy. The aim of our study was to investigate the role of endogenously produced H(2)S in the regulation of mitochondrial electron transport and oxidative phosphorylation in isolated liver mitochondria and in the cultured murine hepatoma cell line Hepa1c1c7. Low concentrations of H(2)S (0.1-1 μM) elicited a significant increase in mitochondrial function, while higher concentrations of H(2)S (3-30 μM) were inhibitory. The positive bioenergetic effect of H(2)S required a basal activity of the Krebs cycle and was most pronounced at intermediate concentrations of succinate. 3-mercaptopyruvate (3-MP), the substrate of the mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) stimulated mitochondrial H(2)S production and enhanced mitochondrial electron transport and cellular bioenergetics at low concentrations (10-100 nM), while at higher concentrations, it inhibited cellular bioenergetics. SiRNA silencing of 3-MST reduced basal bioenergetic parameters and prevented the stimulating effect of 3-MP on mitochondrial bioenergetics. Silencing of sulfide quinone oxidoreductase (SQR) also reduced basal and 3-MP-stimulated bioenergetic parameters. We conclude that an endogenous intramitochondrial H(2)S-producing pathway, governed by 3-MST, complements and balances the bioenergetic role of Krebs cycle-derived electron donors. This pathway may serve a physiological role in the maintenance of mitochondrial electron transport and cellular bioenergetics.
TL;DR: The novel 13,14‐epoxy‐maresin is converted by human macrophages to maresin 1 (MaR1), inhibits leukotriene A4 hydrolase (LTA4H) and shifts macrophage phenotype, and establishes the biosynthesis of the 13 S,14S‐epoxide, its absolute stereochemistry, its precursor role in MaR1 biosynthesis, and its own intrinsic bioactivity.
Abstract: Maresins are produced by macrophages from docosahexaenoic acid (DHA) and exert potent proresolving and tissue homeostatic actions. Maresin 1 (MaR1; 7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid) is the first identified maresin. Here, we investigate formation, stereochemistry, and precursor role of 13,14-epoxy-docosahexaenoic acid, an intermediate in MaR1 biosynthesis. The 14-lipoxygenation of DHA by human macrophage 12-lipoxygenase (hm12-LOX) gave 14-hydro(peroxy)-docosahexaenoic acid (14-HpDHA), as well as several dihydroxy-docosahexaenoic acids, implicating an epoxide intermediate formation by this enzyme. Using a stereo-controlled synthesis, enantiomerically pure 13S,14S-epoxy-docosa-4Z,7Z,9E,11E,16Z,19Z-hexaenoic acid (13S,14S-epoxy-DHA) was prepared, and its stereochemistry was confirmed by NMR spectroscopy. When this 13S,14S-epoxide was incubated with human macrophages, it was converted to MaR1. The synthetic 13S,14S-epoxide inhibited leukotriene B4 (LTB4) formation by human leukotriene A4 hydrolase (LTA4H) ∼40% (P<0.05) to a similar extent as LTA4 (∼50%, P<0.05) but was not converted to MaR1 by this enzyme. 13S,14S-epoxy-DHA also reduced (∼60%; P<0.05) arachidonic acid conversion by hm12-LOX and promoted conversion of M1 macrophages to M2 phenotype, which produced more MaR1 from the epoxide than M1. Together, these findings establish the biosynthesis of the 13S,14S-epoxide, its absolute stereochemistry, its precursor role in MaR1 biosynthesis, and its own intrinsic bioactivity. Given its actions and role in MaR1 biosynthesis, this epoxide is now termed 13,14-epoxy-maresin (13,14-eMaR) and exhibits new mechanisms in resolution of inflammation in its ability to inhibit proinflammatory mediator production by LTA4 hydrolase and to block arachidonate conversion by human 12-LOX rather than merely terminating phagocyte involvement.
TL;DR: It is determined that consuming dietary protein at levels exceeding the RDA may protect fat‐free mass during short‐term weight loss and muscle protein synthesis following weight loss.
Abstract: The purpose of this work was to determine the effects of varying levels of dietary protein on body composition and muscle protein synthesis during energy deficit (ED). A randomized controlled trial of 39 adults assigned the subjects diets providing protein at 0.8 (recommended dietary allowance; RDA), 1.6 (2×-RDA), and 2.4 (3×-RDA) g kg(-1) d(-1) for 31 d. A 10-d weight-maintenance (WM) period was followed by a 21 d, 40% ED. Body composition and postabsorptive and postprandial muscle protein synthesis were assessed during WM (d 9-10) and ED (d 30-31). Volunteers lost (P 0.05) from WM for 2×-RDA and 3×-RDA, but was lower during ED than WM for those consuming RDA levels of protein (energy × protein interaction, P<0.05). To assess muscle protein metabolic responses to varied protein intakes during ED, RDA served as the study control. In summary, we determined that consuming dietary protein at levels exceeding the RDA may protect fat-free mass during short-term weight loss.
TL;DR: A reduction in amplitude of the SCN is sufficient to induce a complete loss of circadian rhythms in energy metabolism and insulin sensitivity, as well as abolishment of normal circadian variation in insulin sensitivity in LL.
Abstract: Circadian rhythm disturbances are observed in, e.g., aging and neurodegenerative diseases and are associated with an increased incidence of obesity and diabetes. We subjected male C57Bl/6J mice to constant light [12-h light-light (LL) cycle] to examine the effects of a disturbed circadian rhythm on energy metabolism and insulin sensitivity. In vivo electrophysiological recordings in the central pacemaker of the suprachiasmatic nuclei (SCN) revealed an immediate reduction in rhythm amplitude, stabilizing at 44% of normal amplitude values after 4 d LL. Food intake was increased (+26%) and energy expenditure decreased (-13%), and we observed immediate body weight gain (d 4: +2.4%, d 14: +5.0%). Mixed model analysis revealed that weight gain developed more rapidly in response to LL as compared to high fat. After 4 wk in LL, the circadian pattern in feeding and energy expenditure was completely lost, despite continuing lowamplitude rhythms in the SCN and in behavior, whereas weight gain had stabilized. Hyperinsulinemic-euglycemic clamp analysis revealed complete abolishment of normal circadian variation in insulin sensitivity in LL. In conclusion, a reduction in amplitude of the SCN, to values previously observed in aged mice, is sufficient to induce a complete loss of circadian rhythms in energy metabolism and insulin sensitivity.
TL;DR: Findings support a central role for TRPA1 and SP in the integration of immune and neuronal mechanisms leading to chronic inflammatory responses and pruritus associated with contact dermatitis.
Abstract: Allergic contact dermatitis is a common skin disease associated with inflammation and persistent pruritus. Transient receptor potential (TRP) ion channels in skin-innervating sensory neurons mediate acute inflammatory and pruritic responses following exogenous stimulation and may contribute to allergic responses. Genetic ablation or pharmacological inhibition of TRPA1, but not TRPV1, inhibited skin edema, keratinocyte hyperplasia, nerve growth, leukocyte infiltration, and antihistamine-resistant scratching behavior in mice exposed to the haptens, oxazolone and urushiol, the contact allergen of poison ivy. Hapten-challenged skin of TRPA1-deficient mice contained diminished levels of inflammatory cytokines, nerve growth factor, and endogenous pruritogens, such as substance P (SP) and serotonin. TRPA1-deficient sensory neurons were defective in SP signaling, and SP-induced scratching behavior was abolished in Trpa1−/− mice. SP receptor antagonists, such as aprepitant inhibited both hapten-induced cutaneous i...
TL;DR: High‐resolution magnetic resonance imaging demonstrated lung damage, edema and consolidation in histone‐injured lungs, confirming the destructive C5a‐dependent effects in lung linked to appearance of extracellular histones.
Abstract: We investigated how complement activation promotes tissue injury and organ dysfunction during acute inflammation. Three models of acute lung injury (ALI) induced by LPS, IgG immune complexes, or C5a were used in C57BL/6 mice, all models requiring availability of both C5a receptors (C5aR and C5L2) for full development of ALI. Ligation of C5aR and C5L2 with C5a triggered the appearance of histones (H3 and H4) in bronchoalveolar lavage fluid (BALF). BALF from humans with ALI contained H4 histone. Histones were absent in control BALF from healthy volunteers. In mice with ALI, in vivo neutralization of H4 with IgG antibody reduced the intensity of ALI. Neutrophil depletion in mice with ALI markedly reduced H4 presence in BALF and was highly protective. The direct lung damaging effects of extracellular histones were demonstrated by airway administration of histones into mice and rats (Sprague-Dawley), which resulted in ALI that was C5a receptor-independent, and associated with intense inflammation, PMN accumulation, damage/destruction of alveolar epithelial cells, together with release into lung of cytokines/chemokines. High-resolution magnetic resonance imaging demonstrated lung damage, edema and consolidation in histone-injured lungs. These studies confirm the destructive C5a-dependent effects in lung linked to appearance of extracellular histones.—Bosmann, M., Grailer, J. J., Ruemmler, R., Russkamp, N. F., Zetoune, F. S., Sarma, J. V., Standiford, T. J., Ward, P. A. Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury.
TL;DR: Brain‐targeted quercetin‐3‐O‐glucuronide may simultaneously modulate multiple independent AD disease‐modifying mechanisms and, as such, may contribute to the benefits of dietary supplementation with red wines as an effective intervention for AD.
Abstract: Epidemiological and preclinical studies indicate that polyphenol intake from moderate consumption of red wines may lower the relative risk for developing Alzheimer's disease (AD) dementia. There is limited information regarding the specific biological activities and cellular and molecular mechanisms by which wine polyphenolic components might modulate AD. We assessed accumulations of polyphenols in the rat brain following oral dosage with a Cabernet Sauvignon red wine and tested brain-targeted polyphenols for potential beneficial AD disease-modifying activities. We identified accumulations of select polyphenolic metabolites in the brain. We demonstrated that, in comparison to vehicle-control treatment, one of the brain-targeted polyphenol metabolites, quercetin-3-O-glucuronide, significantly reduced the generation of β-amyloid (Aβ) peptides by primary neuron cultures generated from the Tg2576 AD mouse model. Another brain-targeted metabolite, malvidin-3-O-glucoside, had no detectable effect on Aβ generation. Moreover, in an in vitro analysis using the photo-induced cross-linking of unmodified proteins (PICUP) technique, we found that quercetin-3-O-glucuronide is also capable of interfering with the initial protein-protein interaction of Aβ1–40 and Aβ1–42 that is necessary for the formation of neurotoxic oligomeric Aβ species. Lastly, we found that quercetin-3-O-glucuronide treatment, compared to vehicle-control treatment, significantly improved AD-type deficits in hippocampal formation basal synaptic transmission and long-term potentiation, possibly through mechanisms involving the activation of the c-Jun N-terminal kinases and the mitogen-activated protein kinase signaling pathways. Brain-targeted quercetin-3-O-glucuronide may simultaneously modulate multiple independent AD disease-modifying mechanisms and, as such, may contribute to the benefits of dietary supplementation with red wines as an effective intervention for AD.—Ho, L., Ferruzzi, M. G., Janle, E. M., Wang, J., Gong, B., Chen, T.-Y., Lobo, J., Cooper, B., Wu, Q. L., Talcott, S. T., Percival, S. S., Simon, J. E., Pasinetti, G. M. Identification of brain-targeted bioactive dietary quercetin-3-O-glucuronide as a novel intervention for Alzheimer's disease.
TL;DR: How Nrf2 functions as a tumor suppressor under normal conditions and how its ability to detoxify the cellular environment makes it an attractive target for other oncogenes either via stabilization or degradation of the transcription factor are focused on.
Abstract: The transcription factor Nrf2 is responsible for regulating a battery of antioxidant and cellular protective genes, primarily in response to oxidative stress. A member of the cap 'n' collar family of transcription factors, Nrf2 activation is tightly controlled by a series of signaling events. These events can be separated into the basal state, a preinduction response, gene induction, and finally a postinduction response, culminating in the restoration of redox homeostasis. However, despite the immensely intricate level of control the cellular environment imposes on Nrf2 activity, there are many opportunities for perturbations to arise in the signaling events that favor carcinogenesis and, therefore, implicate Nrf2 as both a tumor suppressor and a protooncogene. Herein, we highlight the ways in which Nrf2 is regulated, and discuss some of the Nrf2-inducible antioxidant (NQO1, NQO2, HO-1, GCLC), antiapoptotic (Bcl-2), metabolic (G6PD, TKT, PPARγ), and drug efflux transporter (ABCG2, MRP3, MRP4) genes. In addition, we focus on how Nrf2 functions as a tumor suppressor under normal conditions and how its ability to detoxify the cellular environment makes it an attractive target for other oncogenes either via stabilization or degradation of the transcription factor. Finally, we discuss some of the ways in which Nrf2 is being considered as a therapeutic target for cancer treatment.—Shelton, P., Jaiswal, A. K. The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene?
TL;DR: C5L2 has been recently demonstrated to physically interact with both C5aR and β‐arrestin to negatively regulate C 5aR signaling toward an anti‐inflammatory manner, and to reduce pathology, in several disease models in vivo.
Abstract: C5a is the paramount proinflammatory mediator of the complement cascade, and has been previously thought to act only through a single, G-protein-coupled, C5a receptor (C5aR; also termed CD88). In 2000, a second C5a receptor, C5L2 (previously known as GPR77), was discovered; yet, despite 12 yr of intensive research, its biological, or pathophysiological, function is both enigmatic and controversial. Unlike C5aR, this receptor does not couple to G proteins, and early studies promoted the hypothesis that C5L2 functions as a decoy receptor. However, recent data have provided other evidence for more complicated and conflicting interactions between C5L2 and other inflammatory mediators. C5L2 has been recently demonstrated to physically interact with both C5aR and β-arrestin to negatively regulate C5aR signaling toward an anti-inflammatory manner, and to reduce pathology, in several disease models in vivo. In direct contrast, other groups have demonstrated that C5L2 stimulation caused release of HMGB1 both in vitro and in vivo, and enhanced pathology in sepsis models, suggesting a clear proinflammatory signaling role. These astoundingly contradictory data challenge our precepts and complicate the foundational bases for the possible targeting of C5L2 as a therapeutic option in inflammatory disease. C5L2 may be the great masquerader in complement biology; its function dependent on the cell type, species, and disease context. Because of these unusual and unforeseen complexities, we present the current state of knowledge on C5L2 structure, expression and, most controversially, its putative functions.-Li, R., Coulthard, L.G., Wu, M. C. L., Taylor, S. M., Woodruff, T. M. C5L2: a controversial receptor of complement anaphylatoxin, C5a.
TL;DR: It is shown that CD4+ inflammation is a critical regulator of tissue fibrosis and lymphatic dysfunction in lymphedema and that inhibition of Th2 differentiation markedly improves lymphatic function independent of lymphangiogenic cytokine expression.
Abstract: Lymphedema is a dreaded complication of cancer treatment. However, despite the fact that >5 million Americans are affected by this disorder, the development of effective treatments is limited by the fact that the pathology of lymphedema remains unknown. The purpose of these studies was to determine the role of inflammatory responses in lymphedema pathology. Using mouse models of lymphedema, as well as clinical lymphedema specimens, we show that lymphatic stasis results in a CD4 T-cell inflammation and T-helper 2 (Th2) differentiation. Using mice deficient in T cells or CD4 cells, we show that this inflammatory response is necessary for the pathological changes of lymphedema, including fibrosis, adipose deposition, and lymphatic dysfunction. Further, we show that inhibition of Th2 differentiation using interleukin-4 (IL-4) or IL-13 blockade prevents initiation and progression of lymphedema by decreasing tissue fibrosis and significantly improving lymphatic function, independent of lymphangiogenic growth factors. We show that CD4 inflammation is a critical regulator of tissue fibrosis and lymphatic dysfunction in lymphedema and that inhibition of Th2 differentiation markedly improves lymphatic function independent of lymphangiogenic cytokine expression. Notably, preventing and/or reversing the development of pathological tissue changes that occur in lymphedema may be a viable treatment strategy for this disorder.
TL;DR: It is reported that the secreted protein lipocalin 2 (LCN2) amplifies M1 polarization of activated microglia of lipopolysaccharide‐induced mouse neuroinflammation model, suggesting that LCN2 is an M1‐amplifier in brain microglial cells.
Abstract: Activated macrophages are classified into two different forms: classically activated (M1) or alternatively activated (M2) macrophages. The presence of M1/M2 phenotypic polarization has also been suggested for microglia. Here, we report that the secreted protein lipocalin 2 (LCN2) amplifies M1 polarization of activated microglia. LCN2 protein (EC 1 μg/ml), but not glutathione S-transferase used as a control, increased the M1-related gene expression in cultured mouse microglial cells after 8-24 h. LCN2 was secreted from M1-polarized, but not M2-polarized, microglia. LCN2 inhibited phosphorylation of STAT6 in IL-4-stimulated microglia, suggesting LCN2 suppression of the canonical M2 signaling. In the lipopolysaccharide (LPS)-induced mouse neuroinflammation model, the expression of LCN2 was notably increased in microglia. Primary microglial cultures derived from LCN2-deficient mice showed a suppressed M1 response and enhanced M2 response. Mice lacking LCN2 showed a markedly reduced M1-related gene expression in microglia after LPS injection, which was consistent with the results of histological analysis. Neuroinflammation-associated impairment in motor behavior and cognitive function was also attenuated in the LCN2-deficient mice, as determined by the rotarod performance test, fatigue test, open-field test, and object recognition task. These findings suggest that LCN2 is an M1-amplifier in brain microglial cells.
TL;DR: It is demonstrated that ER Ca2+ depletion during the ER stress occurs via translocon, the ER protein complex involved in translation, and suggested that translocon opening is physiologically modulated by GRP78, particularly during theER stress.
Abstract: The endoplasmic reticulum (ER) is involved in many cellular functions, including protein folding and Ca(2+) homeostasis. The ability of cells to respond to the ER stress is critical for cell survival, and disruption in such regulation can lead to apoptosis. ER stress is accompanied by alterations in Ca(2+) homeostasis, and the ER Ca(2+) store depletion by itself can induce ER stress and apoptosis. Despite that, the ER Ca(2+) leak channels activated in response to the ER stress remain poorly characterized. Here we demonstrate that ER Ca(2+) depletion during the ER stress occurs via translocon, the ER protein complex involved in translation. Numerous ER stress inducers stimulate the ER Ca(2+) leak that can be prevented by translocon inhibitor, anisomycin. Expression of GRP78, an ER stress marker, increased following treatment with puromycin (a translocon opener) and was suppressed by anisomycin, confirming a primary role of translocon in ER stress induction. Inhibition of ER store depletion by anisomycin significantly reduces apoptosis stimulated by the ER stress inducers. We suggest that translocon opening is physiologically modulated by GRP78, particularly during the ER stress. The ability to modulate the ER Ca(2+) permeability and subsequent ER stress can lead to development of a novel therapeutic approach.
TL;DR: SGK1‐dependent ion channel regulation may become pathophysiologically relevant primarily after excessive (pathological) expression, and SGK1 may be considered an attractive therapeutic target despite its broad range of functions.
Abstract: The ubiquitously expressed serum- and glucocorticoid-inducible kinase-1 (SGK1) is genomically regulated by cell stress (including cell shrinkage) and several hormones (including gluco- and mineralocorticoids). SGK1 is activated by insulin and growth factors through PI3K and 3-phosphoinositide-dependent kinase PDK1. SGK1 activates a wide variety of ion channels (e.g., ENaC, SCN5A, TRPV4-6, ROMK, Kv1.3, Kv1.5, Kv4.3, KCNE1/KCNQ1, KCNQ4, ASIC1, GluR6, ClCKa/barttin, ClC2, CFTR, and Orai/STIM), which participate in the regulation of transport, hormone release, neuroexcitability, inflammation, cell proliferation, and apoptosis. SGK1-sensitive ion channels participate in the regulation of renal Na+ retention and K+ elimination, blood pressure, gastric acid secretion, cardiac action potential, hemostasis, and neuroexcitability. A common (∼3-5% prevalence in Caucasians and ∼10% in Africans) SGK1 gene variant is associated with increased blood pressure and body weight as well as increased prevalence of type II dia...
TL;DR: Using Cre/LoxP‐based cell lineage tracing in mice, a population of aP2‐expressing progenitors in the stromal vascular fraction (SVF) of both white and brown adipose tissues is identified, adding to the repertoire of adipocyte progenitor markers and points to a new regulator of adipose plasticity.
Abstract: Adipose tissues regulate metabolism, reproduction, and life span. The development and growth of adipose tissue are due to increases of both adipocyte cell size and cell number; the latter is mediated by adipocyte progenitors. Various markers have been used to identify either adipocyte progenitors or mature adipocytes. The fatty acid binding protein 4 (FABP4), commonly known as adipocyte protein 2 (aP2), has been extensively used as a marker for differentiated adipocytes. However, whether aP2 is expressed in adipogenic progenitors is controversial. Using Cre/LoxP-based cell lineage tracing in mice, we have identified a population of aP2-expressing progenitors in the stromal vascular fraction (SVF) of both white and brown adipose tissues. The aP2-lineage progenitors reside in the adipose stem cell niche and express adipocyte progenitor markers, including CD34, Sca1, Dlk1, and PDGFRα. When isolated and grown in culture, the aP2-expressing SVF cells proliferate and differentiate into adipocytes upon induction. Conversely, ablation of the aP2 lineage greatly reduces the adipogenic potential of SVF cells. When grafted into wild-type mice, the aP2-lineage progenitors give rise to adipose depots in recipient mice. Therefore, the expression of aP2 is not limited to mature adipocytes, but also marks a pool of undifferentiated progenitors associated with the vasculature of adipose tissues. Our finding adds to the repertoire of adipose progenitor markers and points to a new regulator of adipose plasticity.
TL;DR: GATOR is identified as a key negative regulator of the Rag GTPases and reveal that, like other mTORC1 regulators, Rag function can be deregulated in cancer.
Abstract: The mTOR complex 1 (mTORC1) pathway promotes cell growth in response to many cues, including amino acids, which act through the Rag guanosine triphosphatases (GTPases) to promote mTORC1 translocation to the lysosomal surface, its site of activation Although progress has been made in identifying positive regulators of the Rags, it is unknown if negative factors also exist Here, we identify GATOR as a complex that interacts with the Rags and is composed of two subcomplexes we call GATOR1 and -2 Inhibition of GATOR1 subunits (DEPDC5, Nprl2, and Nprl3) makes mTORC1 signaling resistant to amino acid deprivation In contrast, inhibition of GATOR2 subunits (Mios, WDR24, WDR59, Seh1L, and Sec13) suppresses mTORC1 signaling, and epistasis analysis shows that GATOR2 negatively regulates DEPDC5 GATOR1 has GTPase-activating protein (GAP) activity for RagA and RagB, and its components are mutated in human cancer In cancer cells with inactivating mutations in GATOR1, mTORC1 is hyperactive and insensitive to amino acid starvation, and such cells are hypersensitive to rapamycin, an mTORC1 inhibitor Thus, we identify a key negative regulator of the Rag GTPases and reveal that, like other mTORC1 regulators, Rag function can be deregulated in cancer
TL;DR: Monocytes/macrophages are of central importance for healing after MI and appear to be an unrecognized new pathophysiological mechanism for left ventricular thrombus development after MI.
Abstract: Myocardial infarction (MI) leads to rapid necrosis of cardiac myocytes. To achieve tissue integrity and function, inflammatory cells are activated, including monocytes/macrophages. However, the effect of monocyte/macrophage recruitment after MI remains poorly defined. After experimental MI, monocytes and macrophages were depleted through serial injections of clodronate-containing liposomes. Monocyte/macrophage infiltration was reduced in the myocardium after MI by active treatment. Mortality was increased due to thromboembolic events in monocyte- and macrophage-depleted animals (92 vs. 33%; P<0.01). Left ventricular thrombi were detectable as early as 24 h after MI; this was reproduced in a genetic model of monocyte/macrophage ablation. A general prothrombotic state, increased infarct expansion, and deficient neovascularization were not observed. Severely compromised extracellular matrix remodeling (collagen I, placebo liposome vs. clodronate liposome, 2.4 ± 0.2 vs. 0.8 ± 0.2 arbitrary units; P<0.001) and locally lost integrity of the endocardium after MI are potential mechanisms. Patients with a left ventricular thrombus had a relative decrease of CD14CD16 monocyte/macrophage subsets in the peripheral blood after MI (no thrombus vs. thrombus, 14.2 ± 0.9 vs. 7.80 ± 0.4%; P<0.05). In summary, monocytes/macrophages are of central importance for healing after MI. Impaired monocyte/macrophage function appears to be an unrecognized new pathophysiological mechanism for left ventricular thrombus development after MI.
TL;DR: Orai3 is established as an ERα‐regulated channel and a potential selective therapeutic target for ERα+ breast cancers and the abrogation of SOCE in MCF7 cells on ERα knockdown can be rescued by ectopic expression of ORAi3.
Abstract: Store-operated Ca(2+) entry (SOCE) encoded by Orai1 proteins is a ubiquitous Ca(2+)-selective conductance involved in cellular proliferation and migration We recently described up-regulation of Orai3 channels that selectively mediate SOCE in estrogen receptor α-expressing (ERα(+)) breast cancer cells However, the connection between ERα and Orai3 and the role of Orai3 in tumorigenesis remain unknown Here, we show that ERα knockdown decreases Orai3 mRNA (by ∼63%) and protein (by ∼44%) with no effect on Orai1 ERα knockdown decreases Orai3-mediated SOCE (by ∼43%) and the corresponding Ca(2+) release-activated Ca(2+) (CRAC) current (by ∼42%) in ERα(+) MCF7 cells The abrogation of SOCE in MCF7 cells on ERα knockdown can be rescued by ectopic expression of Orai3 ERα activation increased Orai3 expression and SOCE in MCF7 cells Epidermal growth factor (EGF) and thrombin stimulate Ca(2+) influx into MCF7 cells through Orai3 Orai3 knockdown inhibited SOCE-dependent phosphorylation of extracellular signal-regulated kinase (ERK1/2; by ∼44%) and focal adhesion kinase (FAK; by ∼46%) as well as transcriptional activity of nuclear factor for activated T cells (NFAT; by ∼49%) Significantly, Orai3 knockdown selectively decreased anchorage-independent growth (by ∼58%) and Matrigel invasion (by ∼44%) of ERα(+) MCF7 cells with no effect on ERα(-) MDA-MB231 cells Moreover, Orai3 knockdown inhibited ERα(+) cell tumorigenesis in immunodeficient mice (∼66% reduction in tumor volume) These data establish Orai3 as an ERα-regulated channel and a potential selective therapeutic target for ERα(+) breast cancers
TL;DR: An improved understanding of the systemic modulations of bile acid metabolism in mammals through the gut‐liver axis is provided.
Abstract: Our understanding of the bile acid metabolism is limited by the fact that previous analyses have primarily focused on a selected few circulating bile acids; the bile acid profiles of the liver and gastrointestinal tract pools are rarely investigated. Here, we determined how chronic ethanol consumption altered the bile acids in multiple body compartments (liver, gastrointestinal tract, and serum) of rats. Rats were fed a modified Lieber-DeCarli liquid diet with 38% of calories as ethanol (the amount equivalent of 4-5 drinks in humans). While conjugated bile acids predominated in the liver (98.3%), duodenum (97.8%), and ileum (89.7%), unconjugated bile acids comprised the largest proportion of measured bile acids in serum (81.2%), the cecum (97.7%), and the rectum (97.5%). In particular, taurine-conjugated bile acids were significantly decreased in the liver and gastrointestinal tract of ethanol-treated rats, while unconjugated and glycine-conjugated species increased. Ethanol consumption caused increased expression of genes involved in bile acid biosynthesis, efflux transport, and reduced expression of genes regulating bile acid influx transport in the liver. These results provide an improved understanding of the systemic modulations of bile acid metabolism in mammals through the gut-liver axis.