Characterization of macroautophagic flux in vivo using a leupeptin-based assay

Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice.     

α9β1 integrin-mediated signaling serves as an intrinsic regulator of pathogenic Th17 cell generation

The interaction between matricellular proteins such as tenascin-C (TN-C) and osteopontin (OPN) and integrins has been implicated in the pathology of rheumatoid arthritis in which Th17 cells are recognized as primary pathogenic cells. The differentiation of Th17 cells is tightly regulated by cytokines derived from APCs, receiving various signals including TLR stimuli. In this study, we used a collagen-induced arthritis model and found that increased numbers of α(9) integrin-positive conventional dendritic cells and macrophage were detectable in the draining lymph node (dLN) shortly following first immunization, and these cells produced both TN-C and OPN, ligands for α(9) integrin. α(9) integrin-mediated signaling, induced by TN-C and OPN, promoted the production of Th17-related cytokines by conventional dendritic cells and macrophages in synergy with TLR2 and 4 signaling. This led to the Th17 cell differentiation and arthritis development. Moreover, Th17 cells generated under blocking of α(9) integrin-mediated signaling showed low level of CCR6 expression and impaired migration ability toward CCL20. Thus, we have identified α(9) integrin-mediated signaling by TN-C and OPN as a novel intrinsic regulator of pathogenic Th17 cell generation that contributes to the development of rheumatoid arthritis.     

Flowering and expression of flowering-related genes under long-day conditions with light-emitting diodes

The effects of light quality on flowering time were investigated in Gypsophila paniculata, which is a long-day cut flower, and with Arabidopsis under long-day conditions with light-emitting diodes (LEDs). Gypsophila paniculata plants were grown under natural daylight and flowering was controlled by long-day treatment with a weak LED light of a single color in the night. Flowering was promoted not by blue light, but by far-red light in G. paniculata, while flowering was promoted by both light colors in Arabidopsis. FT homologs of G. paniculata GpFT1 and GpFT2 were differentially expressed under long-day conditions with white light, suggesting that they play roles in flowering at different stages of reproductive development. GpFTs and FT gene expression was not induced by far-red light in G. paniculata or Arabidopsis. Instead, the expression of the SOC1 homolog of G. paniculata GpSOC1 and SOC1 was induced by far-red light in G. paniculata and Arabidopsis. Flowering was promoted by induction of FT and SOC1 expression with blue light in Arabidopsis, whereas GpFTs and GpSOC1 expression was low with blue light induction in G. paniculata. The relationship between flowering and the expression of FT and SOC1 in Arabidopsis was confirmed with ft and soc1 mutants. These results suggest that long-day conditions with far-red light promote flowering through SOC1 and its homologs, while the conditions with blue light do not promote flowering in G. paniculata, because of low expression of GpFTs and GpSOC1 in contrast to that in Arabidopsis.     

ARD1 binding to RIP1 mediates doxorubicin-induced NF-κB activation

NF-κB is activated by several cellular stresses. Of these, the TNFα-induced activation pathway has been examined in detail. It was recently reported that receptor-interacting protein 1 (RIP1) is involved in DNA damage-induced NF-κB activation by forming a complex with the p53 interacting death domain protein (PIDD) and NF-κB essential modulator (NEMO) in the nucleus, although the underlying mechanism of this interaction has yet to be clarified. This study shows that siRNA knock-down of arrest-defective 1 protein (ARD1) abrogated doxorubicin- but not TNFα-induced activation. Conversely, the over-expression of ARD1 greatly enhanced NF-κB activation induced by doxorubicin. Immunoprecipitation experiments revealed that ARD1 interacted with RIP1 via the acetyltransferase domain. Furthermore, the over-expression of several domain-deleted ARD1 constructs demonstrated that the N-terminal and acetyltransferase domains of ARD1 were required for doxorubicin-induced NF-κB activation. Treatment of deacetylase inhibitor, trichostatin A, significantly increased doxorubicin-induced NF-κB activation in the presence of ARD1 but not acetyltransferase-defective ARD1 mutant. Moreover, N-terminal domain-deleted ARD1 could not be localized in the nucleus in response to doxorubicin treatment. These data indicate that the interaction between ARD1 and RIP1 plays an important role in the DNA damage-induced NF-κB activation, and that the acetyltransferase activity of ARD1 and its localization in to the nucleus are involved in such stress response.     

Molecular characterization of the soybean L-asparaginase gene induced by low temperature stress

L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of the amide group of L-asparagine, releasing aspartate and NH4+. We isolated a low temperature-inducible cDNA sequence encoding L-asparaginase from soybean leaves. The full-length L-asparaginase cDNA, designated GmASP1, contains an open reading frame of 1,258 bp coding for a protein of 326 amino acids. Genomic DNA blotting and fluorescence in situ hybridization showed that the soybean genome has two copies of GmASP1. GmASP1 mRNA was induced by low temperature, ABA and NaCl, but not by heat shock or drought stress. E. coli cells expressing recombinant GmASP1 had 3-fold increased L-asparaginase activity. A possible function of L-asparaginase in the early response to low temperature stress is discussed.     

H1.X with different properties from other linker histones is required for mitotic progression

We report here the characterization of H1.X, a human histone H1 subtype. We demonstrate that H1.X accumulates in the nucleolus during interphase and is distributed at the chromosome periphery during mitosis. In addition, the results of fluorescence recovery after photobleaching indicate that the exchange of H1.X on and off chromatin is faster than that of the other H1 subtypes. Furthermore, RNA interference experiments reveal that H1.X is required for chromosome alignment and segregation. Our results suggest that H1.X has important functions in mitotic progression, which are different from those of the other H1 subtypes.     

Identification of a serum component that regulates cyclooxygenase-2 gene expression in cooperation with 4-hydroxy-2-nonenal

Atherosclerosis is a disorder of lipid metabolism as well as a chronic inflammatory disease. Cyclooxygenase-2 (COX-2), an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses, mediates a variety of biological actions involved in vascular pathophysiology. We have previously shown that COX-2 gene expression is dramatically induced by a lipid-derived endogenous electrophile, 4-hydroxy-2-nonenal (HNE) (Kumagai, T., Matsukawa, N., Kaneko, Y., Kusumi, Y., Mitsumata, M., and Uchida, K. (2004) J. Biol. Chem. 279, 48389-48396). In the present study, based on the finding that HNE induced COX-2 expression only in the serum-containing media, we characterized a serum component essential for the HNE-induced COX-2 induction and found that low density lipoprotein (LDL) that had been denatured by freeze-thawing or oxidized LDL might be involved in the COX-2 induction. Moreover, we characterized the cellular events triggered by the combined stimulus of HNE and oxidized LDL and established that COX-2 induction is regulated by two sets of signaling mechanisms, one for the up-regulation of the scavenger receptor CD36 by HNE and one for the CD36-mediated COX induction by oxidized LDL. These findings represent a demonstration of a link between lipoprotein modification and activation of the inflammatory potential of macrophages.     

Fatty Acids Modulate Toll-like Receptor 4 Activation through Regulation of Receptor Dimerization and Recruitment into Lipid Rafts in a Reactive Oxygen Species-dependent Manner

The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies demonstrated that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4. However, the underlying mechanism has not been understood. Here, we report for the first time that the saturated fatty acid lauric acid induced dimerization and recruitment of TLR4 into lipid rafts, however, dimerization was not observed in non-lipid raft fractions. Similarly, LPS and lauric acid enhanced the association of TLR4 with MD-2 and downstream adaptor molecules, TRIF and MyD88, into lipid rafts leading to the activation of downstream signaling pathways and target gene expression. However, docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, inhibited LPS- or lauric acid-induced dimerization and recruitment of TLR4 into lipid raft fractions. Together, these results demonstrate that lauric acid and DHA reciprocally modulate TLR4 activation by regulation of the dimerization and recruitment of TLR4 into lipid rafts. In addition, we showed that TLR4 recruitment to lipid rafts and dimerization were coupled events mediated at least in part by NADPH oxidase-dependent reactive oxygen species generation. These results provide a new insight in understanding the mechanism by which fatty acids differentially modulate TLR4-mediated signaling pathway and consequent inflammatory responses which are implicated in the development and progression of many chronic diseases.Toll-like receptors (TLRs)³ are one of the key pattern recognition receptor families that play a critical role in inducing innate and adaptive immune responses in mammals by recognizing conserved pathogen-associated molecular pattern of invading microbes. So far, at least thirteen TLRs have been identified in mammalian species (1, 2).Lipopolysaccharide (LPS) from Gram-negative bacteria is the ligand for the TLR4 complex (3), whereas, TLR2 can recognize lipoproteins/lipopeptides of Gram-positive bacteria and mycoplasma (1, 2). LPS forms a complex with LPS-binding protein in serum leading to the conversion of oligomeric micelles of LPS to monomers, which are delivered to CD14. Monomeric LPS is known to bind TLR4/MD-2/CD14 complex (4). Lipid A, which possesses most of the biological activities of LPS, is acylated with hydroxy saturated fatty acids. The 3-hydroxyl groups of these saturated fatty acids are further 3-Ο-acylated by saturated fatty acids. Removal of these Ο-acylated saturated fatty acids from Lipid A not only results in complete loss of endotoxic activity, but also makes Lipid A act as an antagonist against the native Lipid A (5, 6). One or more Lipid As containing unsaturated fatty acids are known to be non-toxic and act as an antagonist against endotoxin (7, 8). In addition, deacylated lipoproteins are unable to activate TLR2 and to induce cytokine expression in monocytes (9). Together, these results suggest that saturated fatty acids acylated on Lipid A or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for TLR4 and TLR2. Indeed, it is suggested that the rapid interaction of bacterial lipopeptides with plasma membrane of macrophages occurs via insertion of their acylated saturated fatty acids as determined by electron energy loss spectroscopy and freeze-fracture techniques (10, 11). TLR2 can form a heterodimer with TLR1 or TLR6, which can discriminate the molecular structure of triacyl or diacyl lipopeptides (12–14). So far there is no evidence that microbial ligands for other TLRs are acylated by saturated fatty acids.Results from our previous studies demonstrated that saturated fatty acids activate TLR4 and polyunsaturated fatty acids (PUFA) inhibit both saturated fatty acid- and LPS-induced activation of TLR4 (15, 16). In addition, the saturated fatty acid lauric acid potentiates, but the n-3 PUFA docosahexaenoic acid (DHA) inhibits lipopeptide (TLR2 agonist)-induced TLR2 activation (17). Together, these results suggest that both TLR2 and TLR4 signaling pathways and target gene expression are reciprocally modulated by saturated and polyunsaturated fatty acids. However, the mechanism for this modulation by fatty acids is not understood.TLR4 is recruited to lipid raft factions after cells are treated with LPS and subsequently induces tumor necrosis factor-α expression in RAW264.7 cells (18). This process occurs in an ROS-dependent manner because inhibition of NADPH oxidase suppresses TLR4 recruitment to lipid rafts (19). Methyl-β-dextrin, a lipid raft inhibitor, significantly inhibits the LPS-induced expression of cytokine (19), suggesting that lipid rafts are essential for TLR4-mediated signal transduction and target gene expression. Lipid rafts are a collection of lipid membrane microdomains characterized by insolubility in non-ionic detergents. Lipid rafts serve as a platform where receptor-mediated signal transduction is initiated (20). Lipid rafts have a special lipid composition that is rich in cholesterol, sphingomyelin, and glycolipids (21). The polar lipids in detergent-resistant membrane contain predominantly saturated fatty acyl residues with underrepresented PUFAs (22–24), suggesting that saturated fatty acyl chains favor lipid raft association. On the other hand, n-3 PUFAs displace signaling proteins from lipid rafts by altering lipid composition, and the displacement leads to the suppression of T-cell receptor-mediated signaling (25). It is now well documented that TLRs form homo- or hetero-oligomers (1, 2). TLR4 homodimerization is the initial step of the receptor activation. Results from our previous studies suggest that the molecular target by which saturated fatty acids and n-3 PUFAs reciprocally modulate TLR4 activation is the receptor complex itself or the event leading to the receptor activation instead of the downstream signaling components (15, 16). Therefore, we determined whether the reciprocal modulation of TLR4 activation is mediated by regulation of the dimerization and recruitment of TLR4 into lipid rafts, and if these processes occur in an ROS-dependent manner.     

INF1 Elicitin Activates Jasmonic Acid- and Ethylene-mediated Signalling Pathways and Induces Resistance to Bacterial Wilt Disease in Tomato

Phytophthora infestans , the cause of potato and tomato late blight disease, produces INF1 elicitin, a 10 kDa extracellular protein. INF1 induces a hypersensitive response (HR) and systemic acquired resistance in species of the Nicotiana genus and a few other genera. We analysed the response of tomato to INF1 and INF1 S3 , which has a Cys to Ser substitution at position 3 of the processed protein and therefore lacks HR induction activity in tobacco. No HR cell death was induced in either INF1- or INF1 S3 -treated tomato leaves. The expression of salicylic acid (SA)-responsive PR-1a ( P6 ) and PR-2a genes was not induced by treatment with either INF1 or INF1 S3 . However, the expression of jasmonic acid (JA)-responsive PR-6 encoding proteinase inhibitor II, LeATL6 encoding ubiquitin ligase E3, and LOX-E encoding lipoxygenase, was up-regulated in tomato leaves treated with INF1 but not in those treated with INF1 S3 . Their induction was completely compromised in INF1-treated jai1-1 mutant tomato, in which the JA signalling pathway is impaired. The accumulation of ethylene (ET) and the expression of ET-responsive genes were also induced in tomato by INF1 but not INF1 S3 treatment. The activation of JA and ET-mediated signals but not the SA-mediated signalling in INF1-treated tomato was also demonstrated by global gene expression analysis. INF1-treated tomatoes, but not those treated with INF1 S3 , exhibited resistance to bacterial wilt disease caused by Ralstonia solanacearum . Thus, INF1 seems to induce resistance to bacterial wilt disease in tomato and activate JA- and ET-mediated signalling pathways without development of HR cell death.