Different from unicellular organisms, metazoan cells require the presence of extracellular growth factors to utilize environmental nutrients. However, the underlying mechanism was unclear. We have delineated a pathway, in which glycogen synthase kinase 3 (GSK3) in cells deprived of growth factors phosphorylates and activates the acetyltransferase KAT5/TIP60, which in turn stimulates the protein kinase ULK1 to elicit autophagy. Cells with the Kat5/Tip60 gene replaced with Kat5S86A that cannot be phosphorylated by GSK3 are resistant to serum starvation-induced autophagy. Acetylation sites on ULK1 were mapped to K162 and K606, and the acetylation-defective mutant ULK1K162,606R displays reduced kinase activity and fails to rescue autophagy in Ulk1−/− mouse embryonic fibroblasts, indicating that acetylation is vital to the activation of ULK1. The GSK3-KAT5-ULK1 cascade seems to be specific for cells to sense growth factors, as KAT5 phosphorylation is not enhanced under glucose deprivation. Distinct from the glucose starvation-autophagy pathway that is conserved in all eukaryotic organisms, the growth factor deprivation response pathway is perhaps unique to metazoan organisms.
GSK3; Tip60; Ulk1; acetylation; autophagy; growth factor; phosphorylation
The fibroblast growth factor (FGF) signaling axis plays important roles in heart development. Yet, the molecular mechanism by which the FGF regulates cardiogenesis is not fully understood. Using genetically engineered mouse and in vitro cultured embryoid body (EB) models, we demonstrate that FGF signaling suppresses premature differentiation of heart progenitor cells, as well as autophagy in outflow tract (OFT) myocardiac cells. The FGF also promotes mesoderm differentiation in embryonic stem cells (ESCs) but inhibits cardiomyocyte differentiation of the mesoderm cells at later stages. Furthermore, inhibition of FGF signaling increases myocardial differentiation and autophagy in both ex vivo cultured embryos and EBs, whereas activation of autophagy promotes myocardial differentiation. Thus, a link between FGF signals preventing premature differentiation of heart progenitor cells and suppression of autophagy has been established. These findings provide the first evidence that autophagy plays a role in heart progenitor differentiation, and suggest a new venue to regulate stem/progenitor cell differentiation.
FGF; autophagy; heart defect; heart development; premature differentiation; second heart field
In this issue of Molecular Cell, Zhang and colleagues describe a critical link between the DNA damage response and the miRNA pathway, in which DNA double strand breaks (DSBs) induce ATM-dependent KSRP phosphorylation to facilitate pri-miRNA processing.
RBX1/ROC1 is an essential subunit of the largest multiunit Cullin-RING E3 ligase (CRL), which controls the degradation of diverse substrates, thereby regulating numerous cellular processes. Recently, we reported that RBX1 is overexpressed in hepatocellular carcinomas (HCC) and its expression is negatively correlated with patient survival. Moreover, siRNA silencing of RBX1 inhibits the proliferation of liver cancer cells both in vitro and in vivo by inducing CDKN1A/p21-dependent cell senescence. Interestingly, independent of senescence, RBX1 knockdown also triggers an autophagy response, due, at least in part, to the accumulation of the MTOR-inhibitory protein DEPTOR, a recently identified CRL substrate. Biologically, blockage of autophagy significantly enhances the growth-suppressive effect of RBX1 knockdown by triggering massive apoptosis, indicating that the autophagy response upon RBX1 knockdown serves as a survival signal in liver cells. Similar observations were also made in many types of human cancer cells upon inhibition of CRL by MLN4924. These findings suggest that RBX1-CRL is a promising anti-cancer drug target and provide proof-of-concept evidence for a novel drug combination of RBX1-CRL inhibitor and autophagy inhibitor for effective treatment of human cancer.
ROC1; RBX1; Cullin-RING E3 ligase; autophagy; senescence; DEPTOR; MTOR; neddylation; MLN4924
The multiunit Cullin (CUL)-RING E3 ligase (CRL) controls diverse biological processes by targeting a mass of substrates for ubiquitination and degradation, whereas its dysfunction causes carcinogenesis. Post-translational neddylation of CUL, a process triggered by the NEDD8-activating enzyme E1 subunit 1 (NAE1), is required for CRL activation. Recently, MLN4924 was discovered via a high-throughput screen as a specific NAE1 inhibitor and first-in-class anticancer drug. By blocking CUL neddylation, MLN4924 inactivates CRL and causes the accumulation of CRL substrates that trigger cell cycle arrest, senescence and/or apoptosis to suppress the growth of cancer cells in vitro and in vivo. Recently, we found that MLN4924 also triggers protective autophagy in response to CRL inactivation. MLN4924-induced autophagy is attributed partially to the inhibition of mechanistic target of rapamycin (also known as mammalian target of rapamycin, MTOR) activity by the accumulation of the MTOR inhibitory protein DEPTOR, as well as reactive oxygen species (ROS)-induced stress. Moreover, the blockage of autophagy response enhances apoptosis in MLN4924-treated cells. Together, our findings not only reveal autophagy as a novel cellular response to CRL inactivation by MLN4924, but also provide a piece of proof-of-concept evidence for the combination of MLN4924 with autophagy inhibitors to enhance therapeutic efficacy.
Cullin-RING E3 ligase; SKP1-Cullin-F-box (SCF) E3 ligase; neddylation; NEDD8-activating enzyme; MLN4924; autophagy; DEPTOR; MTOR
bipolar disorder; classification; depression
A report in this issue of Molecular Cell provides evidence that a translocating SWI/SNF-nucleosome complex efficiently displaces neighboring nucleosomes in vitro and may account for SWI/SNF-dependent nucleosome eviction in vivo.
Accumulating evidence attests to a prosurvival role for autophagy under stress, by facilitating removal of damaged proteins and organelles and recycling basic building blocks, which can be utilized for energy generation and targeted macromolecular synthesis to shore up cellular defenses. These observations are difficult to reconcile with the dichotomous prosurvival and death-inducing roles ascribed to macroautophagy in cardiac ischemia and reperfusion injury, respectively. A careful reexamination of ‘flux’ through the macroautophagy pathway reveals that autophagosome clearance is markedly impaired with reperfusion (reoxygenation) in cardiomyocytes following an ischemic (hypoxic) insult, resulting from reactive oxygen species (ROS)-mediated decline in LAMP2 and increase in BECN1 abundance. This results in impaired autophagy that is ‘ineffective’ in protecting against cell death with ischemia-reperfusion injury. Restoration of autophagosome clearance and by inference, ‘adequate’ autophagy, attenuates reoxygenation-induced cell death.
BECN1; LAMP2; autophagic flux; cell death; ischemia-reperfusion; reactive oxygen species
Proline dehydrogenase (oxidase, PRODH/POX), the first enzyme in the pathway of proline catabolism, has been identified as a mitochondrial, metabolic tumor suppressor, which is downregulated in a variety of human tumors. However, our recent findings show that PRODH/POX is upregulated by hypoxia in vitro and in vivo. The combination of low glucose and hypoxia produces additive effects on PRODH/POX expression. Both hypoxia and glucose depletion enhance PRODH/POX expression through AMP-activated protein kinase (AMPK) activation to promote tumor cell survival. Nevertheless, the mechanisms underlying PRODH/POX prosurvival functions are different for hypoxia and low-glucose conditions. Glucose depletion with or without hypoxia elevates PRODH/POX and proline utilization to supply ATP for cellular energy needs. Interestingly, under hypoxia PRODH/POX induces protective autophagy by generating reactive oxygen species (ROS). AMPK is the main initiator of stress-triggered autophagy. Thus, PRODH/POX acts as a downstream effector of AMPK in the activation of autophagy under hypoxia. This regulation was confirmed to be independent of the mechanistic target of rapamycin (MTOR) pathway, a major downstream target of AMPK signaling.
apoptosis; autophagy; hypoxia; metabolic stress; proline dehydrogenase/oxidase
Osteoarthritis (OA) is the most common form of arthritis and is a major cause of chronic pain and disability. We currently lack disease-modifying OA medical therapeutics that effectively slow or halt the progression to destruction and failure of articular cartilage. Importantly, OA is a disease of the whole joint, including not only meniscal fibrocartilage and hyaline articular cartilage, but also subchondral bone, periarticular musculature, tendons and ligaments, articular adipose tissue, synovium, and synovial fluid (SF). Clinically, varying degrees of synovitis and joint effusion in OA contribute to signs and symptoms of inflammation (1). Multiple lines of evidence suggest that OA progression is promoted by low-grade innate articular inflammation and by synovitis (1,2). “Conventional” inflammatory cytokines expressed in cartilage and synovium likely play a role, and interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), IL-6, IL-8, and IL-17 are among the players in synovitis (1,2). The report by Nair et al in this issue of Arthritis & Rheumatism reveals increased levels of soluble CD14 (sCD14) in SF to be a biomarker of innate inflammation in patients undergoing arthroscopic knee meniscectomy for treatment of meniscal tears (3). Investigators in this group previously characterized this population as “enriched for patients with preradiographic disease” (4), given the associated symptoms, synovitis, and evidence of articular cartilage damage detected by arthroscopy.
Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer, globally. Previously, we showed that Rap1GAP is a tumor suppressor gene that inhibits tumor growth, but promotes invasion in SCCHN. In this work, we discuss the role of Rap1 and Rap1GAP in SCCHN progression in the context of a microRNA-oncogene-tumor suppressor gene axis, and investigate the role of Rap1GAP in EZH2-mediated invasion. Loss of expression of microRNA-101 in SCCHN leads to upregulation of EZH2, a histone methyltransferase. Overexpression of EZH2 silences Rap1GAP via methylation, thereby promoting activation of its target, Rap1. This microRNA-controlled activation of Rap1, via EZH2-mediated silencing of Rap1GAP, is a novel mechanism of Rap1 regulation. In two independent SCCHN cell lines, downregulation of EZH2 inhibits proliferation and invasion. In both cell lines, stable knockdown of EZH2 (shEZH2) recovers Rap1GAP expression and inhibits proliferation. However, siRNA-mediated knockdown of Rap1GAP in these cells rescues proliferation but not invasion. Thus, EZH2 promotes proliferation and invasion via Rap1GAP-dependent and –independent mechanisms, respectively. Although the studies presented here are in the context of SCCHN, our results may have broader implications, given that Rap1GAP acts as a tumor suppressor in pancreatic cancer, thyroid cancer, and melanoma.
EZH2; Rap1GAP; methylation; miR-101; translational
DC play central roles in priming both innate and adaptive immune responses. Multiple DC subsets have been identified on the basis of their phenotype and function. Plasmacytoid DC (pDC) are professional IFN-producing cells that play an essential role in anti-viral immunity. A series of recent studies demonstrates that the regulation of pDC development is different from other types of DC. In this issue of the European Journal of Immunology, new insight is provided into how human pDC development is regulated by various transcription factors, in particular by the Ets family protein Spi-B and E-box protein E2-2.
DC; Developmental immunology; Transcription factors
PI3K; Akt; polycomb group protein; Bmi1; Ezh2; stem cell
The formation of a mature myotendinous junction (MTJ) between a muscle and its site of attachment is a highly regulated process that involves myofiber migration, cell-cell signaling, and culminates with the stable adhesion between the adjacent muscle-tendon cells. Improper establishment or maintenance of muscle-tendon attachment sites results in a decrease in force generation during muscle contraction and progressive muscular dystrophies in vertebrate models. Many studies have demonstrated the important role of the integrins and integrin-associated proteins in the formation and maintenance of the MTJ. We recently demonstrated that moleskin (msk), the gene that encodes for Drosophila importin-7 (DIM-7), is required for the proper formation of muscle-tendon adhesion sites in the developing embryo. Further studies demonstrated an enrichment of DIM-7 to the ends of muscles where the muscles attach to their target tendon cells. Genetic analysis supports a model whereby msk is required in the muscle and signals via the secreted epidermal growth factor receptor (Egfr) ligand Vein to regulate tendon cell maturation. These data demonstrate a novel role for the canonical nuclear import protein DIM-7 in establishment of the MTJ.
Drosophila Importin-7; Egfr signaling; Integrins; myogenesis; myotendinous junction; tendon cells
Obesity has reached epidemic proportions in the United States, and obesity-related illnesses have become a leading preventable cause of death. Childhood obesity is also growing in frequency, and the impact of a lifetime spent in the overweight state is only beginning to emerge in the literature. In this issue of the JCI, Bumaschny et al. used a genetic mouse model to investigate the self-perpetuating nature of obesity and shed some light on why it can become increasingly difficult to lose weight over time.
C. elegans is a model used to study cholesterol metabolism and the functions of its metabolites. Several studies have reported that, in worms, cholesterol is not a structural component of the membrane as it is in vertebrates. However, as in other animals, it is used for the synthesis of steroid hormones that regulate physiological processes such as dauer formation, molting and defecation. After cholesterol is taken up by the gut, mechanisms of transport of cholesterol between tissues in C. elegans involve lipoproteins, as in mammals. A recent study shows that both cholesterol uptake and lipoprotein metabolism in C. elegans are regulated by molecules whose activities, biosynthesis, and secretion strongly resemble those of mammalian bile acids, which are metabolites of cholesterol that act on metabolism in a variety of ways. Importantly, it was found that oxidative stress upsets the regulation of the synthesis of these molecules. Given the known function of mammalian bile acids as metabolic regulators of lipid and glucose homeostasis, future investigations of the biology of C. elegans bile acid-like molecules could provide information on the etiology of human metabolic disorders that are characterized by elevated oxidative stress.
C. elegans; clk-1; bile acids; cholesterol; dafachronic acids; dauer formation; defecation; lipoproteins; metabolic syndrome; oxidative stress; steroid hormones
Powdery mildew pathogens are biotrophic fungi that infect large number of plant species. EDR1 (ENHANCED DISEASE RESISTANCE 1) is a negative regulator of plant disease resistance, and loss-of-function in the EDR1 gene confers enhanced disease resistance to powdery mildew pathogen Golovinomyces cichoracearum. In an edr1 suppressor screen, we recently found that a mutation in HPR1, a component of the THO/TREX complex, suppresses edr1-mediated disease resistance, however the hpr1 mutation enhances the ethylene-induced senescence in edr1. The hpr1 single mutant displays enhanced susceptibility, indicating that HPR1 is involved in plant defense responses.1 THO/TREX is a conserved protein complex that functions in pre-mRNA processing and mRNA export. Several components of THO/TREX complex in Arabidopsis have been identified. By searching Arabidopsis database, we found that Arabidopsis (Columbia-0) has two copies of UAP56, another component of the THO/TREX complex, and the UAP56 proteins are highly conserved. Similar to human UAP56 protein, Arabidopsis UAP56 also localizes to the nucleus, showing a pattern similar to the splicing speckles. Further characterization of the components of THO/TREX in Arabidopsis will provide new insights into the role of THO/TREX in defense responses in plants.
HPR1; THO/TREX complex; UAP56; defense response; powdery mildew
Over the past few years, nitric oxide (NO) has emerged as an important regulator in many physiological events, especially in response to abiotic and biotic stress. However, the roles of NO were mostly derived from pharmacological studies or the mutants impaired NO synthesis unspecifically. In our recent study, we highlighted a novel strategy by expressing the rat neuronal NO synthase (nNOS) in Arabidopsis to explore the in vivo role of NO. Our results suggested that plants were able to perform well in the constitutive presence of nNOS, and provided a new class of plant experimental system with specific in vivo NO release. Furthermore, our findings also confirmed that the in vivo NO is essential for most of environmental abiotic stresses and disease resistance against pathogen infection. Proper level of NO may be necessary and beneficial, not only in plant response to the environmental abiotic stress, but also to biotic stress.
abiotic and biotic stress; disease resistance; drought; in vivo; nitric oxide; nitric oxide synthase; salinity
SUMOylation of transcription factors and chromatin proteins is in many cases a negative mark that recruits factors that repress gene expression. In this study, we determined the occupancy of Small Ubiquitin-like MOdifier (SUMO)-1 on chromatin in HeLa cells by use of chromatin affinity purification coupled with next-generation sequencing. We found SUMO-1 localization on chromatin was dynamic throughout the cell cycle. Surprisingly, we observed that from G1 through late S phase, but not during mitosis, SUMO-1 marks the chromatin just upstream of the transcription start site on many of the most active housekeeping genes, including genes encoding translation factors and ribosomal subunit proteins. Moreover, we found that SUMO-1 distribution on promoters was correlated with H3K4me3, another general chromatin activation mark. Depletion of SUMO-1 resulted in downregulation of the genes that were marked by SUMO-1 at their promoters during interphase, supporting the concept that the marking of promoters by SUMO-1 is associated with transcriptional activation of genes involved in ribosome biosynthesis and in the protein translation process.
We analyzed modification of chromatin by ubiquitination in human cells and whether this mark changes through the cell cycle. HeLa cells were synchronized at different stages and regions of the genome with ubiquitinated chromatin were identified by affinity purification coupled with next-generation sequencing. During interphase, ubiquitin marked the chromatin on the transcribed regions of ∼70% of highly active genes and deposition of this mark was sensitive to transcriptional inhibition. Promoters of nearly half of the active genes were highly ubiquitinated specifically during mitosis. The ubiquitination at the coding regions in interphase but not at promoters during mitosis was enriched for ubH2B and dependent on the presence of RNF20. Ubiquitin labeling of both promoters during mitosis and transcribed regions during interphase, correlated with active histone marks H3K4me3 and H3K36me3 but not a repressive histone modification, H3K27me3. The high level of ubiquitination at the promoter chromatin during mitosis was transient and was removed within 2 h after the cells exited mitosis and entered the next cell cycle. These results reveal that the ubiquitination of promoter chromatin during mitosis is a bookmark identifying active genes during chromosomal condensation in mitosis, and we suggest that this process facilitates transcriptional reactivation post-mitosis.
The dynamic remodeling of actin filaments in guard cells functions in stomatal movement regulation. In our previous study, we found that the stochastic dynamics of guard cell actin filaments play a role in chloroplast movement during stomatal movement. In our present study, we further found that tubular actin filaments were present in tobacco guard cells that express GFP-mouse talin; approximately 2.3 tubular structures per cell with a diameter and height in the range of 1–3 µm and 3–5 µm, respectively. Most of the tubular structures were found to be localized in the cytoplasm near the inner walls of the guard cells. Moreover, the tubular actin filaments altered their localization slowly in the guard cells of static stoma, but showed obvious remodeling, such as breakdown and re-formation, in moving guard cells. Tubular actin filaments were further found to be colocalized with the chloroplasts in guard cells, but their roles in stomatal movement regulation requires further investigation.
actin dynamics; tubular actin filaments; chloroplast; guard cell; stomatal movement
To identify biological and clinical predictors of acute kidney injury in subjects with acute lung injury.
Secondary data analysis from a multicenter, randomized clinical trial.
Intensive care units in ten university medical centers.
A total of 876 patients enrolled in the first National Heart, Lung, and Blood Institute Acute Respiratory Distress Syndrome Clinical Network trial.
Study subjects were randomized to receive a low tidal volume ventilation strategy and pharmacologic therapy with ketoconazole or lisofylline in a factorial design.
Measurements and Main Results
We tested the association of baseline levels of interleukin-6, interleukin-8, interleukin-10, von Willebrand factor, tumor necrosis factor-α, type I and II soluble tumor necrosis factor receptors (sTNFR-I and -II), protein C, plasminogen activator inhibitor-1 (PAI-1), surfactant protein-A, surfactant protein-D, and intracellular adhesion molecule-1 with subsequent acute kidney injury. Of 876 study participants who did not have end-stage renal disease, 209 (24%) developed acute kidney injury, defined as a rise in serum creatinine of >50% from baseline over the first four study days. The 180-day mortality rate for subjects with acute kidney injury was 58%, compared with 28% in those without acute kidney injury (p < .001). Interleukin-6, sTNFR-I, sTNFR-II, and PAI-1 levels were independently associated with acute kidney injury after adjustment for demographics, interventions, and severity of illness. A combination of clinical and biological predictors had the best area under the receiver operating characteristic curve, and the contribution of sTNFR-I and PAI-1 to this model was highly significant (p = .0003).
Elevations in PAI-1, interleukin-6, and the sTNFRs in subjects with acute kidney injury suggest that disordered coagulation, inflammation, and neutrophil–endothelial interactions play important roles in the pathogenesis of acute kidney injury. The combination of these biological and clinical risk factors may have important and additive value in predictive models for acute kidney injury.
acute kidney injury; acute lung injury; acute respiratory distress syndrome; biological marker; predictive value; interleukin-6; soluble tumor necrosis factor receptor; plasminogen activator inhibitor-1
Arabidopsis mutant hps1 that over-accumulates sucrose has enhanced sensitivity in almost all the aspects of plant responses to phosphate starvation. The detailed characterization of hps1 has led to the conclusion that sucrose is a global regulator of plant phosphate responses. Here, we show that hps1 is also hypersensitive to nitrogen and potassium deprivation, as well as to decreased levels of overall macronutrients. These results suggest that sucrose regulates plant deficiency responses to multiple nutrients and is part of a general response to nutrient deprivation.
nutrient deficiency; plant response; hps1; sucrose; signaling